CN114073996B - Nested micro-well array chip and preparation method thereof - Google Patents
Nested micro-well array chip and preparation method thereof Download PDFInfo
- Publication number
- CN114073996B CN114073996B CN202111404997.3A CN202111404997A CN114073996B CN 114073996 B CN114073996 B CN 114073996B CN 202111404997 A CN202111404997 A CN 202111404997A CN 114073996 B CN114073996 B CN 114073996B
- Authority
- CN
- China
- Prior art keywords
- microwell
- micro
- cell
- well
- array
- 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.)
- Active
Links
Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/10—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by centrifugation ; Cyclones
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Sustainable Development (AREA)
- Biomedical Technology (AREA)
- Dispersion Chemistry (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Clinical Laboratory Science (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Hematology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Fluid Mechanics (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention provides a nested addressable microwell array chip, which comprises microwell units distributed in an array, wherein the microwell units comprise microwells distributed in an array, each microwell comprises a cell capturing microwell and a cell culture microwell which is arranged above the cell capturing microwell and communicated with the cell capturing microwell, the size of the cell culture microwell is larger than that of the cell capturing microwell, and a code is arranged on each microwell unit. The invention also provides a preparation method of the nested addressable micro-well array chip. The chip provided by the invention does not influence the single cell capture rate, and simultaneously provides a larger cell culture space, thereby being beneficial to further culture of cells. Meanwhile, each micro-well unit is provided with a code, so that accurate tracking and observation of single cells can be realized.
Description
Technical Field
The invention belongs to the technical field of microfluidic chips, and particularly relates to a nested addressable microwell array chip, a die and a preparation method of the die.
Background
In recent years, in research related to single cell analysis, a main high-throughput single cell analysis platform is a microfluidic chip, i.e., a single cell is separated and captured by using micron-sized functional elements such as micro-channels, micro-pores or micro-electrodes integrated on the chip, and then is cultured and tracked on the chip. The high-throughput analysis is helpful for obtaining single cell information with statistical significance, and has an important role in determining that the difference result is from random fluctuation or has special biological function significance.
The single cell capturing and culturing method based on the micro-fluidic chip mainly comprises the following steps: a micro-well array method, a micro-droplet method, a hydraulic capture method and an external field control method. The microwell array method is widely applied because of the advantages of convenient operation, simple chip design, no need of introducing a large number of flow channel designs, high flux and the like. In the prior art, the size of a microwell in a microwell array method is close to the size of a cell so as to improve the single cell capture rate by using the size exclusion principle, but the narrow space of the microwell limits the growth and migration of the cell and is not beneficial to the long-term culture and observation of the single cell.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a nested addressable microwell array chip, a mold and a method for manufacturing the same, which can achieve both single cell capture rate and culture space.
In order to achieve the above objects, the present invention provides a nested addressable microwell array chip, which comprises microwell units distributed in an array, wherein the microwell units comprise microwells distributed in an array, the microwells comprise cell capture microwells and cell culture microwells disposed on the cell capture microwells and communicated with the cell capture microwells, the size of the cell culture microwells is larger than that of the cell capture microwells, and the microwell units are provided with codes.
In one embodiment, the microwell unit code is located in the center of the microwell unit.
In one embodiment, the cell culture microwells have a height of 20-30 μm and the cell capture microwells have a height of 20-30 μm.
In one embodiment, the perpendicular bisector of the cell capture microwell cross-section and the perpendicular bisector of the cell culture microwell cross-section coincide.
In one embodiment, the cell-capture microwell is circular in cross-section and 15-30 μm in diameter; the cross section of the cell culture micro-well is square, and the side length is 80-120 mu m.
In one embodiment, the nested addressable microwell array chip comprises ten rows and ten columns of microwell units, and the interval between adjacent microwell units is 200-220 μm.
In one embodiment, the microwell unit comprises five rows and five columns of microwells, and the centers of adjacent microwells are spaced 170-190 μm apart.
In one embodiment, the code is disposed in the third row and third column of the microwell unit.
The invention also provides a preparation method of the nested addressable micro-well array chip, which comprises the following steps:
providing a mold, wherein the mold comprises a substrate and microstructure array units which are arranged on the substrate and distributed in an array, the microstructure array units comprise microstructures distributed in an array, and the microstructures comprise cell culture micro-well female molds arranged on the substrate and cell capture micro-well female molds arranged on the cell culture micro-well female molds; the size of the cell culture micro-well female die is larger than that of the cell capture micro-well female die; and the microstructure array unit is provided with a coding female die.
And adding a chip forming material into the die, and demolding after forming to obtain the chip.
In one embodiment, the chip forming material is polydimethylsiloxane prepolymer, and the weight ratio of the component A to the component B is 5-15.
The invention also provides a mold, which comprises a substrate and microstructure array units which are arranged on the substrate and distributed in an array manner, wherein the microstructure array units comprise microstructures distributed in an array manner, and each microstructure comprises a cell culture micro-well female die arranged on the substrate and a cell capture micro-well female die arranged on the cell culture micro-well female die; the size of the negative mould of the cell culture micro-well is larger than that of the negative mould of the cell capture micro-well; and the microstructure array unit is provided with a coding female die.
The chip provided by the invention comprises microwell units distributed in an array, wherein the microwell units comprise microwells distributed in an array, and each microwell comprises a cell capturing microwell and a cell culture microwell which is arranged on the cell capturing microwell and is communicated with the cell capturing microwell; the cell culture microwells are larger in size than the cell capture microwells; and the micro-well unit is provided with a code. The chip provided by the invention can realize high-throughput culture of single cells, and is provided with the cell culture micro-well and the cell capture micro-well, wherein the size of the cell culture micro-well is larger than that of the cell capture micro-well, and the cell culture micro-well is arranged above the cell capture micro-well, so that the single cell capture rate is not influenced, meanwhile, a larger culture space can be provided for the cells, and the further culture of the cells is facilitated. In addition, each micro-well unit is provided with a code, so that accurate tracking observation of single cells can be realized. The nested addressable micro-well array chip provided by the invention can be applied to single-cell capture, culture and observation, and has wide application prospects in the fields of life sciences, medicine, cell biology and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a schematic structural diagram of a nested addressable micro-well array chip according to an embodiment of the present invention;
FIG. 2 is a top view micrograph of a nested addressable microwell array chip microwell unit, with a 100 μm scale, according to an embodiment of the present invention;
FIG. 3 is a cross-sectional micrograph of a nested addressable microwell array chip, with a 100 μm ruler, according to an embodiment of the present invention;
FIG. 4 is a schematic diagram of a process for capturing single cells by using a nested addressable micro-well array chip according to an embodiment of the present invention;
FIG. 5 is a micrograph of fluorescent microspheres captured by a nested addressable microwell array chip, with a 100 μm scale, according to an embodiment of the present invention;
FIG. 6 is a micrograph of a single cell captured by a nested addressable microwell array chip provided in an embodiment of the present invention, wherein the left image is a micrograph of an FDA/PI stained living cell, and the right image is a micrograph of an FDA/PI stained dead cell in the same region, with a scale of 100 μm;
FIG. 7 is a micrograph of a nested addressable microwell array chip for single cell culture according to an embodiment of the present invention.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. However, the present invention is not limited to the following embodiments, and all other embodiments obtained by those skilled in the art without any inventive work are within the scope of the present invention.
The invention provides a nested addressable micro-well array chip, which comprises micro-well units distributed in an array, wherein the micro-well units comprise micro-wells distributed in an array, and each micro-well comprises a cell capturing micro-well and a cell culture micro-well which is arranged on the cell capturing micro-well and is communicated with the cell capturing micro-well; the cell culture microwells have a size larger than the cell capture microwells; and the micro-well unit is provided with a code.
Referring to fig. 1, fig. 2 and fig. 3, fig. 1 is a schematic structural view of a nested addressable microwell array chip provided by an embodiment, fig. 2 is a top-view micrograph of a microwell unit of the nested addressable microwell array chip provided by the embodiment, fig. 3 is a cross-sectional micrograph of the nested addressable microwell array chip provided by the embodiment, in which fig. 11 represents a microwell unit, 111 represents a microwell, 112 represents a code, 1111 represents a cell culture microwell, and 1112 represents a cell capture microwell.
The nested addressable microwell array chip comprises microwell units 11, the microwell units 11 are distributed in an array, each microwell unit 11 comprises microwells 111 distributed in an array, each microwell 111 comprises a cell culture microwell 1111 and a cell capture microwell 1112, the cell culture microwell size 1111 is larger than the cell capture microwell size 1112, and the microwell units 11 are provided with codes 112.
In one embodiment, the cell culture microwells 1111 have a height of 20-30 μm, preferably 25-30 μm, more preferably 25 μm, and may have a square cross-section with sides of 80-120 μm, preferably 90-110 μm, more preferably 100 μm; the cell-capture microwells 1112 are 20-30 μm, preferably 22-25 μm, and more preferably 25 μm in height, and may be circular in cross-section and 15-30 μm, preferably 22-25 μm, and more preferably 25 μm in diameter.
In one embodiment, the perpendicular bisector of the cross-section of cell capture microwell 1112 and the perpendicular bisector of the cross-section of cell culture microwell 1111 coincide, i.e., the cell culture microwell 1111 is directly above the cell capture microwell 1112, such that cells are captured by the cell capture microwell through the cell culture microwell.
In one embodiment, one microwell unit 11 may comprise five rows and five columns of microwells 111, with centers of adjacent microwells 111 spaced 170-190 μm apart, preferably 170-180 μm apart, and more preferably 180 μm apart.
The microwell unit 11 of the present invention is provided with a code 112, and the code 112 can be disposed at any position of the microwell unit 11, preferably at the center of the microwell unit 11. In one embodiment, the microwell unit 11 includes five rows and five columns of microwells 111, and the code 112 may be disposed in the third row and the third column of the microwell unit 11. In the present invention, the code may be a digital code, for example, starting from 00, which is encoded in the arrangement of the microwell units.
The microwell units 11 of the present invention are distributed in an array on the nested addressable microwell array chip, and in one embodiment, the microwell units 11 may comprise ten rows and ten columns, and the interval between adjacent microwell units is 200-220 μm, preferably 200-210 μm, and more preferably 210 μm.
In one embodiment of the present invention, the chip comprises ten rows and ten columns of microwell units, each microwell array comprises five rows and five columns, wherein the third row and the third column are digitally encoded, and the other 24 positions are microwells, at this time, the whole chip can be observed in the same observation field under the microscope objective lens of 10 times, and the microwell units and microwells in the microwell units can be accurately encoded, identified and tracked. For example, in a microwell unit containing the numerical code 00, microwells located in the first row and the second column have the code 00-A2, as shown in FIG. 1. Therefore, in the chip provided by the application, the single cells captured by each nested microwell can be coded and identified one by one, and the problem that the high-density and periodically arranged microwells in the traditional microwell culture chip and the single cells captured by the microwell culture chip are easy to confuse and miss addresses because no special mark exists, so that all the single cells are difficult to accurately track is solved.
The number of the microwell array, the center interval of the adjacent microwells, the number of the microwell array and the interval of the adjacent microwell units are related to the size of the nested addressable microwell array chip. In one embodiment, the nested addressable microwell array chip comprises ten rows and ten columns of microwell units, the interval between adjacent microwell units is 200-220 μm, each microwell unit comprises five rows and five columns of microwells, and the center interval between adjacent microwells is 170-190 μm. In other embodiments, the number of microwell arrays, the center spacing between adjacent microwells, the number of microwell unit arrays and the spacing between adjacent microwell units can be adjusted by one skilled in the art to achieve the observation in the same field of view, which is not limited by the present invention.
The chip provided by the invention can be used for capturing single cells, after the chip is mixed with a single cell suspension and centrifuged, cells can be captured by both the upper layer cell culture micro-well and the lower layer cell capture micro-well, but the cells in the upper layer cell culture micro-well are easier to wash away than the cells in the lower layer cell capture micro-well, so the cells in the upper layer cell culture micro-well are washed away after multiple times of solution washing, and the cells in the lower layer cell capture micro-well are retained. The method solves the problem that the traditional single-layer microwell reduces the size of the microwell to improve the single cell capture rate so as to limit the cell growth, and simultaneously overcomes the defect that the single cell capture efficiency is low because a dilute cell suspension is used for avoiding simultaneously capturing a plurality of single cells in the microwell when a large-size microwell is used. In one embodiment, single cell suspension with concentration of 300,000/mL is used, and the single cell capture rate of the chip is as high as 35.0 +/-18.2%, which is 44 times of the capture rate (0.8%) of the traditional single-layer large microwell using natural deposition.
The invention also provides a mold, which comprises a substrate and microstructure array units which are arranged on the substrate and distributed in an array manner, wherein the microstructure array units comprise microstructures distributed in an array manner, and each microstructure comprises a cell culture micro-well female die arranged on the substrate and a cell capture micro-well female die arranged on the cell culture micro-well female die; the size of the cell culture micro-well female die is larger than that of the cell capture micro-well female die; and the microstructure array unit is provided with a coding female die.
The mold provided by the invention is used for preparing the nested addressable micro-well array chip in the technical scheme, the structure and the size of the mold are suitable for the nested addressable micro-well array chip in the technical scheme, and the details are not repeated.
In the present invention, the mold may be prepared in the following manner:
forming a first layer of photoresist on a substrate, covering a first mask, and carrying out first ultraviolet exposure to obtain a first layer of photoresist with a potential pattern;
forming a second layer of photoresist on the first layer of photoresist with the potential pattern, covering a second mask, and carrying out second ultraviolet exposure to obtain a second layer of photoresist with the potential pattern;
and developing the two layers of photoresist with the potential patterns on the substrate, and performing post-treatment to obtain the mold.
The invention firstly forms a first layer of photoresist on a substrate for preparing a cell culture micro-well female die. In the invention, the substrate can be a silicon wafer, and the first layer of photoresist can be a negative photoresist. The invention can form a first layer of photoresist on a substrate by adopting a spin coating mode, and the spin coating parameters are as follows: spin at 500rpm for 12s followed by 3000rpm for 30s.
After the first layer of photoresist is formed, prebaking is carried out, and a solvent is removed; covering a first reticle, the reticle having a pattern associated with the photoresist. In the invention, the photoresist is a negative photoresist, and the pattern part of the mask is exposed; covering the first mask, and then carrying out ultraviolet exposure, wherein the photoresist on the pattern part of the first mask is crosslinked, and the photoresist which is not crosslinked is subsequently removed; and (3) carrying out postbaking after ultraviolet exposure to obtain a first layer of photoresist with a potential pattern, wherein the first potential pattern is a pattern comprising a cell culture micro-well array. The first mask can be prepared according to the following method:
drawing a plane graph of a chip comprising microwell units distributed in an array by using AutoCAD, wherein the microwell units comprise cell culture microwells distributed in an array;
the planar pattern is made into a first mask.
And forming a second layer of photoresist on the first layer of photoresist with the potential pattern for preparing a cell capture micro-well female die. In the present invention, the second layer of photoresist may be a negative photoresist. The invention can form the second layer of photoresist by adopting a spin coating mode, and the spin coating parameters are as follows: spin at 500rpm for 12s followed by 3000rpm for 30s.
And after the second layer of photoresist is formed, pre-baking is carried out, a mask is covered, ultraviolet exposure is carried out, and post-baking is carried out, so that the second layer of photoresist with a potential pattern is obtained, wherein the second potential pattern is the pattern comprising the cell capture microwell array. The second mask can be prepared according to the following method:
drawing a plane graph of a chip comprising microwell units distributed in an array by using AutoCAD, wherein the microwell units comprise cell capture microwells distributed in an array;
the planar pattern is made into a second mask.
And developing the two layers of photoresist with the potential patterns on the substrate by using a developing solution, and removing the photoresist which is not crosslinked to obtain the photoresist with the two layers of patterns.
After the development treatment, post-treatment is performed. The post-treatment specifically comprises the following steps: and treating the photoresist of the two-layer pattern with a silanization reagent so as to eliminate surface active groups and obtain the photoresist mould.
The invention also provides a preparation method of the nested addressable micro-well array chip, which comprises the following steps:
providing a mould;
and adding a chip forming material into the die, and demolding after forming to obtain the chip.
In the process of preparing the nested addressable micro-well array chip, the mold is the mold described in the above technical scheme or the mold prepared according to the method described in the above technical scheme, and the details are not repeated herein.
And after obtaining the die, adding a chip forming material into the die, and demolding after forming to obtain the chip.
In the invention, the adopted chip forming material can be polydimethylsiloxane prepolymer and comprises a component A and a component B. In one embodiment, the weight ratio of the polydimethylsiloxane prepolymer component a to the component B is 5.
After the nested addressable micro-well array chip is obtained, the embedded addressable micro-well array chip is sterilized, and the method specifically comprises the following steps: and placing the nested addressable micro-well array chip in a high-temperature steam boiler for high-temperature sterilization, and storing in an aseptic environment.
In one embodiment, the method further comprises cutting the chip into individual chips of 1cm × 1cm size before sterilizing the chip.
The chip provided by the invention can be used for capturing cells and culturing cells, and referring to fig. 4, fig. 4 is a schematic flow chart of a nested addressable microwell array chip for capturing single cells provided by an embodiment.
The invention takes the fluorescent microspheres as a cell model to carry out a microsphere capture experiment, and the capture rate of the chip provided by the invention to the fluorescent microspheres is 64.8 +/-12.6%; when the chip provided by the invention is used for capturing single cells, the single cell capturing efficiency is 35.0 +/-18.2%, and the single cell capturing rate is different from the highest single microsphere capturing rate because the size difference between cells is larger than that of fluorescent microspheres; after the single cell capture is carried out by adopting the chip provided by the invention, the measured single cell survival rate is 98.3 +/-3.4 percent, which shows that the method is safe to the cells; and (3) culturing after capturing the single cells, and observing that the single cells gradually migrate out of the lower layer cell capturing microwells and enter the upper layer cell culturing microwells for normal growth and proliferation.
The following describes the nested addressable micro-well array chip, the mold and the preparation method thereof provided by the present invention with reference to the following examples.
Example 1:
preparing a nested addressable micro-well array chip according to the following steps:
manufacturing a photoetching mask, comprising the following steps: drawing a plane graph corresponding to a first layer of square array by using AutoCAD, wherein the side length of each square is 100 micrometers, the interval between the centers of adjacent squares is 180 micrometers, each 24 squares form a square array unit, the centers of the square array units draw numbers in sequence, and the adjacent square arrays are 210 micrometers apart; drawing ten rows and ten columns of square matrix units in the area range of 1cm multiplied by 1cm, wherein 100 square matrix units are contained; drawing a plane graph corresponding to the second layer of circular array by using AutoCAD according to the relative coordinates of the first layer of square array graph, wherein the graph parameters are as follows: the diameter of the circle is 20 μm, the interval between the centers of adjacent squares is 180 μm, each 24 circles form a group to form a square matrix unit, the centers of the square matrix units draw numbers in sequence, and the adjacent square matrix units are 210 μm apart; drawing ten rows and ten columns of square matrix units in the area range of 1cm multiplied by 1cm, wherein 100 square matrix units are contained; making a plane graph corresponding to the first layer of square array into a first mask; and manufacturing a second mask plate by using a plane pattern corresponding to the second layer of addressable circular array.
Preparing a photoresist mold, comprising the following steps: taking a clean silicon wafer as a substrate, and spin-coating a first layer of SU-8 3050 photoresist on the substrate, wherein the spin-coating parameters are as follows: spin coating at 500rpm for 12s, and then at 3000rpm for 30s; and then carrying out prebaking, wherein the prebaking parameters are as follows: heating at 65 deg.C for 1min, and heating at 95 deg.C for 12min; placing the first mask plate above the SU-8 3050 photoresist layer, and performing ultraviolet exposure with an exposure machine for 18s at an exposure power of 9.5mW/cm 2 And then carrying out postbaking, wherein postbaking parameters are as follows: heating at 65 deg.C for 1min, and heating at 95 deg.C for 3min; spin-coating a second layer of SU-8 2025 photoresist on the first layer of SU-8 3050 photoresist, wherein the spin-coating parameters are as follows: spin-coating at 500rpm for 12s, followed by spin-coating at 3000rpm for 30s; carrying out prebaking, wherein the prebaking parameters are as follows: heating at 65 deg.C for 1min, and heating at 95 deg.C for 10min; placing the second mask above the second SU-8 2025 photoresist layer, and performing ultraviolet alignment exposure with an exposure machine with exposure time of 15s and exposure power of 9.5mW/cm 2 (ii) a Post-exposure is carried out for 18s with an exposure power of 9.5mW/cm 2 And then carrying out postbaking, wherein postbaking parameters are as follows: at a temperature of 65 DEG CHeating for 1min, and heating at 95 deg.C for 3min to obtain double-layer photoresist with potential pattern; processing the double-layer photoresist with the potential patterns on the substrate for 3min by using a developing solution and then developing to obtain the photoresist with the double-layer patterns; the photoresist having the double layer pattern is treated with a silylating agent to obtain a mold.
The preparation method of the nested addressable micro-well array chip comprises the following steps: mixing a polydimethylsiloxane prepolymer component A and a component B according to a weight ratio of 10 to 1, and pouring the mixture on a photoresist mould; air is pumped by a vacuum pump for about 30min to remove bubbles; standing the prepolymer mixture after removing bubbles, and standing overnight at normal temperature to solidify the prepolymer mixture; removing the cured polydimethylsiloxane chip from the die, and cutting the whole polydimethylsiloxane chip into independent chips with the size of 1cm multiplied by 1cm to obtain a nested addressable micro-well array chip; placing the nested addressable micro-well array chip in a high-temperature steam pot for high-temperature sterilization; and (4) placing the sterilized chip in an aseptic environment for storage.
Examples 2 to 4:
the diameter is 15 mu m, and the density is 1.08g/cm 3 The fluorescent microsphere simulates cells to carry out a capture experiment:
putting the nested addressable micro-well array chip provided by the embodiment 1 into a centrifuge tube containing a PDMS pile body; 5mL of the solution was added at a concentration of 3X 10 5 Centrifuging a/mL fluorescent microsphere suspension, a nested addressable microwell array chip and a centrifuge tube at the centrifugal speed of 600g for 1s; taking out the chip after centrifugation is finished, and washing to remove redundant fluorescent microspheres on the chip; referring to fig. 5, the single fluorescent microsphere capture rate of the chip is calculated to be 64.8 ± 12.6%, wherein the capture efficiency is calculated according to formula (I):
capture efficiency = number of microwells per array containing a single fluorescent microsphere/24 x 100%. And replacing the centrifugal rotation speed of 600g with 300g, and calculating to obtain the fluorescent microsphere with the capture rate of 1.6 +/-2.1%.
And replacing the centrifugal rotation speed of 600g with 450g, and calculating to obtain the fluorescent microsphere capture rate of 21.3 +/-9.8%.
Example 5:
example 1 is mentionedPutting the chip into a sterile centrifuge tube containing a PDMS pile body; 5mL of the solution was added at a concentration of 3X 10 5 Centrifuging a single cell suspension of/mL, a nested addressable microwell array chip and a centrifuge tube at the centrifugal rotation speed of 600g for 1s; taking out the chip after the centrifugation is finished, and washing the chip by using a PBS buffer solution to remove redundant cells; the single cell capture rate of the chip was calculated to be 35.0. + -. 18.2%.
Determining the survival rate of single cells, and determining cell death and viability by utilizing a Fluorescein Diacetate (FDA)/Propidium Iodide (PI) staining experiment, wherein the final concentration of FDA is 2 mug/mL, the final concentration of PI is 10 mug/mL, and the staining time is 30min; referring to fig. 6, the survival rate of the captured single cells was calculated to be 98.3 ± 3.4%, indicating that the method is safe for cells.
Performing single cell culture, placing the chip for capturing single cells in a living cell workstation for culture, and performing continuous observation; tracking and shooting the microwells initially capturing the single cells at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 18h, 24h, 36h, 48h, 60h, 72h, 84h and 96h respectively; referring to fig. 7, it can be observed that the cells are normal in morphology and can normally proliferate, while it is observed that single cells will gradually migrate out of the lower cell capture microwells into the upper cell culture microwells.
The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, it is possible to make various improvements and modifications to the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (9)
1. A nested addressable microwell array chip, comprising microwell units distributed in an array, wherein the microwell units comprise microwells distributed in an array, and the microwells comprise cell capture microwells and cell culture microwells arranged on the cell capture microwells and communicated with the cell capture microwells;
the cell culture microwells are larger in size than the cell capture microwells;
the micro-well unit is provided with a code;
the cell culture micro-well is provided with a cell suspension inlet and a cell culture solution outlet;
the perpendicular bisector of the cross-section of the cell capture microwell coincides with the perpendicular bisector of the cross-section of the cell culture microwell.
2. The nested addressable microwell array chip of claim 1, wherein the code is located in the center of the microwell unit.
3. The nested addressable microwell array chip of claim 1, wherein the height of the cell culture microwells in the microwells is 20-30 μm and the height of the cell capture microwells is 20-30 μm.
4. The nested addressable microwell array chip of claim 3, wherein the cell-trapping microwells are circular in cross-section and 15-30 μm in diameter;
the cross section of the cell culture micro-well is square, and the side length is 80-120 mu m.
5. The nested addressable microwell array chip of claim 4, wherein the nested addressable microwell array chip comprises ten rows and ten columns of microwell units, adjacent microwell units being spaced 200-220 μ ι η apart;
each micro-well unit comprises five rows and five columns of micro-wells, and the centers of the adjacent micro-wells are spaced from 170 to 190 mu m.
6. The nested addressable microwell array chip of claim 5, wherein the coding is disposed in the microwell unit at the position of the third row and the third column.
7. The method for preparing the nested addressable micro-well array chip of any one of claims 1 to 6, comprising the following steps:
providing a mold, wherein the mold comprises a substrate and microstructure array units which are arranged on the substrate and distributed in an array, the microstructure array units comprise microstructures distributed in an array, and the microstructures comprise a cell culture micro-well female die arranged on the substrate and a cell capture micro-well female die arranged on the cell culture micro-well female die; the size of the negative mould of the cell culture micro-well is larger than that of the negative mould of the cell capture micro-well; the microstructure array unit is provided with a coding female die; a cell suspension inlet and a cell culture solution outlet are formed in the cell culture micro-well female die;
the perpendicular bisector of the cross section of the cell capturing micro-well female die is coincided with the perpendicular bisector of the cross section of the cell culture micro-well female die;
and adding a chip forming material into the mold, and demolding after forming to obtain the chip.
8. The method of claim 7, wherein the die-forming material is polydimethylsiloxane prepolymer.
9. A mold is characterized by comprising a substrate and microstructure array units which are arranged on the substrate and distributed in an array, wherein the microstructure array units comprise microstructures distributed in an array, and the microstructures comprise cell culture micro-well female molds arranged on the substrate and cell capture micro-well female molds arranged on the cell culture micro-well female molds;
the size of the cell culture micro-well female die is larger than that of the cell capture micro-well female die;
the microstructure array unit is provided with a coding female die;
the cell culture micro-well female die is provided with a cell suspension inlet and a cell culture solution outlet;
the perpendicular bisector of the cross section of the cell capturing micro-well female die is coincident with the perpendicular bisector of the cross section of the cell culturing micro-well female die.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111404997.3A CN114073996B (en) | 2021-11-24 | 2021-11-24 | Nested micro-well array chip and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111404997.3A CN114073996B (en) | 2021-11-24 | 2021-11-24 | Nested micro-well array chip and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114073996A CN114073996A (en) | 2022-02-22 |
| CN114073996B true CN114073996B (en) | 2023-04-14 |
Family
ID=80284635
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111404997.3A Active CN114073996B (en) | 2021-11-24 | 2021-11-24 | Nested micro-well array chip and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114073996B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115318351B (en) * | 2022-08-29 | 2024-02-23 | 中山大学·深圳 | Single-cell paired double-layer micro-well array chip and preparation method and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5948363A (en) * | 1996-04-22 | 1999-09-07 | Gaillard; Patrick | Micro-well strip with print tabs |
| CN104884605A (en) * | 2012-08-24 | 2015-09-02 | 耶鲁大学 | System, device and method for high-throughput multi-plexed detection |
| KR20160134329A (en) * | 2015-05-15 | 2016-11-23 | 성균관대학교산학협력단 | Microfluidic chip for screening cancer drug resistance cell and use thereof |
| WO2018051242A1 (en) * | 2016-09-14 | 2018-03-22 | Ecole Polytechnique Federale De Lausanne (Epfl) | Device for high throughput single-cell studies |
| CN108611250A (en) * | 2018-05-10 | 2018-10-02 | 北京纳米能源与系统研究所 | A kind of biochip and preparation method thereof of unicellular positioning and screening based on micro-nano spherical cavity array |
| KR20210104244A (en) * | 2020-02-17 | 2021-08-25 | 중앙대학교 산학협력단 | Vertically Coated Graphene Oxide Micro-well plate, preparation method thereof and Drug screening method for cancer treatment using same |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AR066170A1 (en) * | 2007-09-26 | 2009-07-29 | Genentech Inc | ANTI INTEGRINA ALFA 5 BETA 1 |
| TW201137121A (en) * | 2010-04-26 | 2011-11-01 | Univ Nat Changhua Education | Cell culture real-time observation system |
| TWI588256B (en) * | 2015-11-20 | 2017-06-21 | 財團法人國家衛生研究院 | Device and method for single cell isolation and cultivation |
| CN106047706B (en) * | 2016-06-15 | 2018-11-16 | 西北工业大学 | One kind realizing cellular localization culture chip and its use and preparation method based on unicellular capture |
| KR102127241B1 (en) * | 2017-05-12 | 2020-06-26 | 서울대학교산학협력단 | Method of isolation, retrieval, analysis and recovery of cells and cell secretions using microstructures |
| CN109926110B (en) * | 2019-03-26 | 2021-08-24 | 上海天马微电子有限公司 | Chip substrate and microfluidic chip |
| CN117264765A (en) * | 2019-05-31 | 2023-12-22 | 西安医学院 | Cell capturing and tumor ball culturing array chip and preparation and operation method thereof |
-
2021
- 2021-11-24 CN CN202111404997.3A patent/CN114073996B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5948363A (en) * | 1996-04-22 | 1999-09-07 | Gaillard; Patrick | Micro-well strip with print tabs |
| CN104884605A (en) * | 2012-08-24 | 2015-09-02 | 耶鲁大学 | System, device and method for high-throughput multi-plexed detection |
| KR20160134329A (en) * | 2015-05-15 | 2016-11-23 | 성균관대학교산학협력단 | Microfluidic chip for screening cancer drug resistance cell and use thereof |
| WO2018051242A1 (en) * | 2016-09-14 | 2018-03-22 | Ecole Polytechnique Federale De Lausanne (Epfl) | Device for high throughput single-cell studies |
| CN108611250A (en) * | 2018-05-10 | 2018-10-02 | 北京纳米能源与系统研究所 | A kind of biochip and preparation method thereof of unicellular positioning and screening based on micro-nano spherical cavity array |
| KR20210104244A (en) * | 2020-02-17 | 2021-08-25 | 중앙대학교 산학협력단 | Vertically Coated Graphene Oxide Micro-well plate, preparation method thereof and Drug screening method for cancer treatment using same |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114073996A (en) | 2022-02-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105861309B (en) | A kind of super-hydrophobic micro-pit array chip and preparation method and application | |
| CN101629143B (en) | Microfluidic cell array chip for high-throughput medicament screening, method and use | |
| CN101245311B (en) | Three-dimensional high-flux medicaments sifting chip and manufacture method thereof | |
| CN102337213B (en) | Polydimethylsiloxane (PDMS)-based three-dimensional single cell culture chip and controllable preparation method thereof | |
| CN114073996B (en) | Nested micro-well array chip and preparation method thereof | |
| Jeong et al. | “Living” microvascular stamp for patterning of functional neovessels; orchestrated control of matrix property and geometry | |
| CN103421691B (en) | Glass chip for cultivating single cell array based on microfluidic patterning technology and preparation method thereof | |
| CN103122311B (en) | Flexible three-dimensional single-cell targeted cultivating chip and controllable preparation method thereof | |
| CN109234163B (en) | High-throughput tumor targeted drug concentration screening microfluidic device | |
| WO2010074659A1 (en) | 3d mold for manufacture of sub-micron 3d structures using 2-d photon lithography and nanoimprinting and process thereof | |
| CN110551681B (en) | Micro-fluidic chip for simulating embryo implantation angiogenesis and preparation method and application thereof | |
| CN100425420C (en) | Method of producing resin molded product | |
| KR102157266B1 (en) | Perimysium-scaled heart on a chip and uses thereof | |
| CN104053459A (en) | Method and device for constructing three-dimensional cellular microenvironment on the basis of transparent sponge scaffold | |
| CN106479893B (en) | Device and method for patterning co-culture of multiple cells | |
| CN110257249A (en) | A kind of micro-fluidic chip and administration cultural method for tumour cell dimensional culture | |
| CN204079986U (en) | A kind of micro-nano-fluidic control device for cell migration research | |
| CN113083383B (en) | Microfluidic chip device, preparation method and soil microbial community culture method | |
| JP5753148B2 (en) | Method for culturing spheroids using cell culture carrier and cell culture carrier | |
| CN110862926A (en) | Multilayer paper chip based on microfluidic technology and construction method thereof | |
| CN105176817B (en) | Self-circulation cell bioreactor based on alternate-current electric heating and manufacturing method and using method thereof | |
| CN115318351B (en) | Single-cell paired double-layer micro-well array chip and preparation method and application thereof | |
| CN103360459A (en) | Constant current diffused protein crystallization method, open type constant current diffused protein crystallization array chip and manufacture method and application of chip | |
| Qiao et al. | High throughput and uniform tumour spheroids culture system for drug screening | |
| CN103992948A (en) | Micronanofluidic device for researching cell migration |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |