CN114073996B - A nested microwell array chip and its preparation method - Google Patents

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

A nested microwell array chip and its preparation method

technical field

The invention belongs to the technical field of microfluidic chips, and in particular relates to a nested addressable microwell array chip, a mold and a preparation method thereof.

Background technique

In recent years, in the research related to single-cell analysis, the main high-throughput single-cell analysis platform is the microfluidic chip, that is, the single-cell Isolated and captured, cultured and tracked on a chip. High-throughput analysis helps to obtain statistically significant single-cell information, and plays an important role in determining whether differential results are due to random fluctuations or have special biological function significance.

Single cell capture and culture methods based on microfluidic chips mainly include: microwell array method, micro droplet method, hydraulic capture method and external field manipulation method. Among them, the microwell array method is widely used because of its convenient operation, simple chip design, no need to introduce a large number of flow channel designs, and high throughput. In the prior art, in the microwell array method, the size of the microwells is close to the size of the cells, and the principle of size exclusion is used to improve the capture rate of single cells. However, the narrow space of the microwells limits the growth and migration of cells, which is not conducive to the long-term cultivation and development of single cells. observe.

Contents of the invention

In view of this, the technical problem to be solved by the present invention is to provide a nested addressable microwell array chip, a mold and a preparation method thereof, which take into account the capture rate of single cells and the culture space.

In order to achieve the above object, the present invention provides a nested addressable micro-well array chip, the nested addressable micro-well array chip includes micro-well units distributed in arrays, and the micro-well units include array-distributed The microwells include cell trapping microwells and cell culture microwells arranged on the cell trapping microwells and communicated with the cell trapping microwells, and the size of the cell culture microwells is larger than that of the cells Captures the dimensions of the microwells on which the codes are provided.

In one embodiment, the micro-well unit code is located at the center of the micro-well unit.

In one embodiment, the height of the cell culture microwell is 20-30 μm, and the height of the cell capture microwell is 20-30 μm.

In one embodiment, the perpendicular line of the cross section of the cell trapping microwell coincides with the perpendicular line of the cross section of the cell culture microwell.

In one embodiment, the cell capture microwell has a circular cross section with a diameter of 15-30 μm; the cell culture microwell has a square cross section with a side length of 80-120 μm.

In one embodiment, the nested addressable microwell array chip includes microwell units in ten rows and ten columns, and the interval between adjacent microwell units is 200-220 μm.

In one embodiment, the microwell unit includes microwells in five rows and five columns, and the distance between the centers of adjacent microwells is 170-190 μm.

In one embodiment, the code is set at the position of the third row and the third column of the microwell unit.

The present invention also provides a method for preparing a nested addressable microwell array chip, comprising:

A mold is provided, the mold includes a substrate and a microstructure array unit arranged on the substrate and distributed in an array, the microstructure array unit includes a microstructure distributed in an array, and the microstructure includes a microstructure arranged on the substrate The cell culture microwell negative mold and the cell capture microwell negative mold arranged on the cell culture microwell negative mold; the size of the cell culture microwell negative mold is greater than the size of the cell capture microwell negative mold; the A coded negative mold is arranged on the microstructure array unit.

Adding chip forming material into the mold, demoulding after molding to obtain the chip.

In one embodiment, the chip forming material is polydimethylsiloxane prepolymer, and the weight ratio of component A to component B is 5:1-15:1.

The present invention also provides a mold, including a substrate and a microstructure array unit arranged on the substrate and distributed in an array, the microstructure array unit includes a microstructure distributed in an array, and the microstructure includes a microstructure arranged on the substrate The cell culture microwell negative mold on the substrate and the cell capture microwell negative mold arranged on the cell culture microwell negative mold; the size of the cell culture microwell negative mold is larger than the size of the cell capture microwell negative mold ; The microstructure array unit is provided with a coded negative mold.

The chip provided by the present invention includes a microwell unit distributed in an array, and the microwell unit includes a microwell distributed in an array, and the microwell includes a cell capture microwell and is arranged on the cell capture microwell and connected to the cell capture microwell. Cell culture microwells with wells connected; the size of the cell culture microwells is larger than the size of the cell capture microwells; codes are set on the microwell units. The chip provided by the present invention can realize the high-throughput culture of single cells. The chip is provided with cell culture microwells and cell capture microwells, wherein the size of the cell culture microwells is greater than the size of the cell capture microwells, and the cell culture microwells are set at Above the cell capture microwell, it will not affect the single cell capture rate, and at the same time, it can provide a large culture space for cells, which is conducive to the further cultivation of cells. In addition, each microwell unit is provided with a code, which can realize accurate tracking and observation of single cells. The nested addressable microwell array chip provided by the invention can be applied to single cell capture, culture and observation, and has broad application prospects in the fields of life science, medicine, cell biology and the like.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only It is an embodiment of the present invention, and those skilled in the art can also obtain other drawings according to the provided drawings without creative work.

FIG. 1 is a schematic structural view of a nested addressable microwell array chip provided by an embodiment of the present invention;

Fig. 2 is a top view micrograph of the nested addressable microwell array chip microwell unit provided by the embodiment of the present invention, with a scale of 100 μm;

Figure 3 is a cross-sectional micrograph of the nested addressable microwell array chip provided by the embodiment of the present invention, the scale bar is 100 μm;

Fig. 4 is a schematic flow diagram of capturing single cells by a nested addressable microwell array chip provided by an embodiment of the present invention;

Fig. 5 is a micrograph of fluorescent microspheres captured by the nested addressable microwell array chip provided by the embodiment of the present invention, the scale bar is 100 μm;

Figure 6 is a microscopic picture of a single cell captured by a nested addressable microwell array chip provided by an embodiment of the present invention. The left picture in the figure is a microscopic picture of living cells stained by FDA/PI, and the right picture is FDA in the same area. / Micrographs of PI-stained dead cells, scale bar 100 μm;

Fig. 7 is a micrograph of single cell culture on the nested addressable microwell array chip provided by the embodiment of the present invention.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. But the present invention is not limited to the following embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work all belong to the protection scope of the present invention.

The present invention provides a nested addressable micro-well array chip, which comprises a micro-well unit distributed in an array. A cell culture microwell on the cell capture microwell and communicated with the cell capture microwell; the size of the cell culture microwell is larger than that of the cell capture microwell; codes are set on the microwell unit.

Referring to Figure 1, Figure 2 and Figure 3, Figure 1 is a schematic structural diagram of the nested addressable microwell array chip provided by the embodiment, and Figure 2 is the microwell unit of the nested addressable microwell array chip provided by the embodiment Figure 3 is a cross-sectional micrograph of the nested addressable microwell array chip provided by the embodiment, in which 11 represents the microwell unit, 111 represents the microwell, 112 represents the code, and 1111 represents the cell Culture microwells, 1112 indicate cell capture microwells.

The nested addressable microwell array chip described in the present invention includes microwell units 11, and the microwell units 11 are distributed in an array, and each microwell unit 11 includes microwells 111 distributed in an array, and the microwells 111 include cell culture Microwell 1111 and cell capture microwell 1112 , cell culture microwell size 1111 is larger than cell capture microwell size 1112 , and microwell unit 11 is provided with code 112 .

In one embodiment, the height of the cell culture microwell 1111 is 20-30 μm, preferably 25-30 μm, more preferably 25 μm, and its cross section can be a square with a side length of 80-120 μm, preferably 90-110 μm, More preferably 100 μm; the height of the cell capture microwell 1112 is 20-30 μm, preferably 22-25 μm, more preferably 25 μm, its cross section can be circular, and its diameter is 15-30 μm, preferably 22-25 μm, more Preferably it is 25 μm.

In one embodiment, the mid-perpendicular line of the cross-section of the cell trapping microwell 1112 coincides with the mid-perpendicular line of the cross-section of the cell culture microwell 1111, that is, the cell culture microwell 1111 is directly above the cell trapping microwell 1112, In order to facilitate cells passing through the cell culture microwells to be captured by the cell capture microwells.

In one embodiment, one microwell unit 11 may include microwells 111 in five rows and five columns, and the distance between adjacent microwells 111 is 170-190 μm, preferably 170-180 μm, more preferably 180 μm.

In the present invention, the micro-well unit 11 is provided with a code 112, and the code 112 can be set at any position of the micro-well unit 11, preferably at the center of the micro-well unit 11. In one embodiment, the microwell unit 11 includes microwells 111 in five rows and five columns, and the code 112 can be set at the position of the third row and the third column of the microwell unit 11 . In the present invention, the code can be a digital code, for example, starting from 00, and coded according to the arrangement of microwell units.

In the present invention, microwell units 11 are distributed in an array on the nested addressable microwell array chip. In one embodiment, microwell units 11 in ten rows and ten columns may be included, and the interval between adjacent microwell units is 200-220 μm, preferably 200-210 μm, more preferably 210 μm.

In one embodiment of the present invention, the chip includes ten rows and ten columns of micro-well units, and each micro-well array includes an array of five rows and five columns, wherein the third row and the third column are digital codes, and the other 24 positions are Microwells, at this time, the entire chip can be observed in the same observation field of view under the 10x objective lens of the microscope, and the microwell units and the microwells in the microwell units can be accurately coded, identified and tracked. For example, in the microwell unit with digital code 00, the microwell located in the first row and second column has code 00-A2, as shown in Fig. 1 . Therefore, in the chip provided by this application, each single cell captured by nested microwells can also be encoded and identified one by one, which solves the problem of high-density, periodically arranged microwells and their captured single cells in traditional microwell culture chips. Cells are easily confused and lost without special markings, so it is difficult to achieve accurate tracking of all single cells.

In the present invention, the number of microwell arrays, the distance between adjacent microwell centers, the number of microwell unit arrays, and the distance between adjacent microwell units are related to the size of the nested addressable microwell array chip. In one embodiment, the nested addressable microwell array chip includes ten rows and ten columns of microwell units, the interval between adjacent microwell units is 200-220 μm, and each microwell unit includes five rows and five columns of microwells, The distance between the centers of adjacent microwells is 170-190 μm. In other embodiments, in order to achieve observation in the same field of view, those skilled in the art can adjust the number of microwell arrays, the distance between adjacent microwell centers, the number of microwell unit arrays and the distance between adjacent microwell units. There are no special restrictions.

The chip provided by the present invention can be used for single cell capture. After it is mixed with single cell suspension and centrifuged, both the upper layer cell culture microwell and the lower layer cell capture microwell can capture cells, but the cells in the upper layer cell culture microwell are larger than the cells in the lower layer. The cells in the trapping microwells are more likely to be washed away, so the cells in the upper cell culture microwells will be washed away after repeated washing with solution, while the cells in the lower cell trapping microwells are retained. This method solves the problem that the traditional single-layer microwell reduces the size of the microwell to increase the capture rate of single cells, thereby limiting the growth of cells, and also overcomes the problem of using large-sized microwells to avoid capturing multiple single cells in the microwell at the same time. The use of dilute cell suspension results in the disadvantage of low capture efficiency of single cells. In one embodiment, using a single cell suspension with a concentration of 300,000/mL, the single cell capture rate of the chip is as high as 35.0 ± 18.2%, which is the capture rate (~0.8%) of the traditional single-layer large and micro wells using natural deposition. 44 times.

The present invention also provides a mold, including a substrate and a microstructure array unit arranged on the substrate and distributed in an array, the microstructure array unit includes a microstructure distributed in an array, and the microstructure includes a microstructure arranged on the substrate The cell culture microwell negative mold on the substrate and the cell capture microwell negative mold arranged on the cell culture microwell negative mold; the size of the cell culture microwell negative mold is larger than the size of the cell capture microwell negative mold ; The microstructure array unit is provided with a coded negative mold.

The mold provided by the present invention is used to prepare the nested addressable microwell array chip described in the above technical solution, and the structure and size of the mold are compatible with the nested addressable microwell array chip described in the above technical solution. The invention will not be described in detail.

In the present invention, the mold can be prepared in the following manner:

forming a first layer of photoresist on the substrate, covering the first mask, and performing the first ultraviolet exposure to obtain the first layer of photoresist with potential patterns;

Forming a second layer of photoresist on the first layer of photoresist with latent patterns, covering the second mask, and performing a second UV exposure to obtain the second layer of photoresist with latent patterns;

The two layers of photoresist with latent patterns on the substrate are developed, and the mold is obtained after post-processing.

In the invention, the first layer of photoresist is firstly formed on the substrate, which is used to prepare the negative mold of the cell culture microwell. In the present invention, the substrate can be a silicon wafer, and the first layer of photoresist can be a negative photoresist. In the present invention, the first layer of photoresist can be formed on the substrate by means of spin coating, and the spin coating parameters are: spin coating at 500 rpm for 12 seconds, and then spin coating at 3000 rpm for 30 seconds.

After the first layer of photoresist is formed, prebaking is performed to remove the solvent; the first mask is covered, and the pattern of the mask is related to the photoresist. In the present invention, the photoresist is a negative photoresist, and the pattern part of the mask plate is exposed; after covering the first mask plate, ultraviolet exposure is performed, and the photoresist of the pattern part of the first mask plate is cross-linked , the non-crosslinked photoresist is subsequently removed; post-baking is performed after ultraviolet exposure to obtain a first layer of photoresist with a latent pattern, and the first latent pattern is a pattern including a cell culture microwell array. Wherein, the first mask plate can be prepared according to the following method:

Utilize AutoCAD to draw the planar figure of the chip that comprises the micro-well unit that is distributed in array, and described micro-well unit comprises the cell culture micro-well that is distributed in array;

The above-mentioned planar figure is made into a first mask plate.

A second layer of photoresist is formed on the first layer of photoresist with latent patterns, which is used to prepare the negative mold of the cell trapping microwell. In the present invention, the second layer of photoresist can be a negative photoresist. In the present invention, the second layer of photoresist can be formed by spin coating, and the spin coating parameters are: spin coating at 500 rpm for 12 seconds, and then spin coating at 3000 rpm for 30 seconds.

After forming the second layer of photoresist, perform pre-baking, cover the mask plate, perform ultraviolet exposure, and post-baking to obtain the second layer of photoresist with potential patterns. The second potential pattern is the array of cell trapping microwells graphics. Wherein, the second mask plate can be prepared according to the following method:

Utilize AutoCAD to draw the planar figure of the chip that comprises the micro-well unit that is distributed in array, and described micro-well unit comprises the cell capture micro-well that is distributed in array;

The above-mentioned planar figure is made into a second mask plate.

The two layers of photoresist with latent patterns on the substrate are developed with a developer to remove the photoresist that has not been cross-linked to obtain a photoresist with two layers of patterns.

After the development process, post-processing is performed. The post-treatment specifically includes: treating the photoresist of the two-layer pattern with a silylating agent, so as to eliminate surface active groups, and obtain a photoresist mold.

The present invention also provides a method for preparing a nested addressable microwell array chip, comprising:

Provide molds;

Adding chip forming material into the mold, demoulding after molding to obtain the chip.

In the process of preparing a nested addressable microwell array chip, the mold is the mold described in the above technical solution or a mold prepared according to the method described in the above technical solution, and the present invention will not repeat them here.

After the mold is obtained, the chip forming material is added into the mold, and the chip is demoulded after molding to obtain the chip.

In the present invention, the chip forming material used may be polydimethylsiloxane prepolymer, including component A and component B. In one embodiment, the weight ratio of polydimethylsiloxane prepolymer component A to component B is 5:1-15:1, preferably 5:1-10:1, more preferably 10:1 1. After adding polydimethylsiloxane prepolymer to the mold, remove air bubbles, let it stand overnight at room temperature, separate the mold after curing, and obtain a nested addressable microwell array chip.

After the nested addressable micro-well array chip is obtained, it is sterilized, specifically: the nested addressable micro-well array chip is placed in a high-temperature steam pot for high-temperature sterilization, and stored in a sterile environment .

In one embodiment, before sterilizing the chip, it further includes cutting the whole polydimethylsiloxane chip into independent chips with a size of 1 cm×1 cm.

The chip provided by the present invention can be used to capture cells and culture cells, see Figure 4, Figure 4 is a schematic flow chart of the nested addressable microwell array chip provided in the embodiment to capture single cells, the chip is placed in a single cell suspension In the process, under the action of centrifugation, the single cells enter the cell culture microwell and the cell capture microwell, and are washed with PBS buffer. cell capture.

The present invention uses fluorescent microspheres as a cell model to carry out microsphere capture experiments, and the chip provided by the present invention has a capture rate of fluorescent microspheres of 64.8 ± 12.6%; using the chip provided by the present invention for single cell capture, the single cell capture efficiency is 35.0% ±18.2%, there is a difference between the single cell capture rate and the highest single microsphere capture rate, the reason is that the size difference between cells is greater than that of fluorescent microspheres; after using the chip provided by the invention for single cell capture, the measured single cell viability rate is 98.3 ±3.4%, indicating that this method is safe for cells; after capturing single cells and culturing, it was observed that single cells gradually migrated out of the lower layer cell capture microwells and entered the upper layer cell culture microwells for normal growth and proliferation.

The nested addressable microwell array chip, the mold and the preparation method thereof provided by the present invention will be further described below in conjunction with the examples.

Example 1:

Follow the steps below to fabricate a nested addressable microwell array chip:

Making a photolithography mask includes the following steps: use AutoCAD to draw the plane figure corresponding to the square array on the first layer, the side length of the square is 100 μm, the distance between the centers of adjacent squares is 180 μm, and every 24 squares form a square array Units, numbers are drawn in order in the center of the square matrix unit, and the adjacent square matrix is separated by 210 μm; a total of ten rows and ten columns of square matrix units are drawn within the area of 1cm×1cm, which contains a total of 100 square matrix units; according to the first layer of square array For the relative coordinates of the graphics, use AutoCAD to draw the plane graphics corresponding to the second layer of circular arrays. The graphics parameters are: the diameter of the circle is 20 μm, the distance between the centers of adjacent squares is 180 μm, and every group of 24 circles forms a square. Array unit, the center of the square matrix unit draws numbers in order, and the distance between adjacent square matrix units is 210 μm; draw a total of ten rows and ten columns of square matrix units within the area of 1cm×1cm, containing a total of 100 square matrix units; the first layer The planar pattern corresponding to the square array is made as the first mask; the planar pattern corresponding to the addressable circular array of the second layer is made as the second mask.

Prepare a photoresist mold, including the following steps: use a clean silicon wafer as a substrate, spin-coat the first layer of SU-8 3050 photoresist on the substrate, and the spin-coating parameters are: spin-coat at a speed of 500rpm for 12s, and then spin-coat at a speed of 3000rpm Spin coating for 30s; then pre-baking, the pre-baking parameters are: heating at 65°C for 1min, and then heating at 95°C for 12min; place the first mask on the SU-8 3050 photoresist layer, use exposure The UV exposure time is 18s, the exposure power is 9.5mW/cm ² , and then post-baking. The post-baking parameters are: heating at 65°C for 1min, then heating at 95°C for 3min; The second layer of SU-8 2025 photoresist was spin-coated on the SU-8 3050 photoresist. The spin-coating parameters were: spin-coating at a speed of 500rpm for 12s, and then spin-coating at a speed of 3000rpm for 30s; pre-baking, pre-baking parameters: at Heating at 65°C for 1min, and then heating at 95°C for 10min; place the second mask on top of the second layer of SU-8 2025 photoresist layer, and use an exposure machine to perform UV alignment exposure with an exposure time of 15s. The exposure power is 9.5mW/cm ² ; after exposure, the exposure time is 18s, the exposure power is 9.5mW/cm ² , and then post-baking, the post-baking parameters are: heating at 65°C for 1min, then heating at 95°C Under heating for 3 minutes, a double-layer photoresist with a latent pattern is obtained; the photoresist with a double-layer latent pattern on the substrate is treated with a developer for 3 minutes and then developed to obtain a photoresist with a double-layer pattern; it is treated with a silylating agent A photoresist with a double-layer pattern, resulting in a mold.

The preparation of a nested addressable microwell array chip comprises the following steps: mixing polydimethylsiloxane prepolymer component A and component B according to a weight ratio of 10:1, and pouring them on a photoresist mold; Use a vacuum pump to pump air for about 30 minutes to remove air bubbles; leave the prepolymer mixture after removing air bubbles to stand, and place it overnight at room temperature to solidify; remove the cured polydimethylsiloxane chip from the mold, Cut the whole polydimethylsiloxane chip into independent chips with a size of 1cm×1cm to obtain a nested addressable microwell array chip; place the nested addressable microwell array chip in a high-temperature steam pot Sterilize at high temperature; store the sterilized chip in a sterile environment.

Embodiment 2～4:

The capture experiment was carried out with fluorescent microspheres with a diameter of 15 μm and a density of 1.08 g/cm ³ simulating cells:

Put the nested addressable microwell array chip provided in Example 1 into a centrifuge tube containing PDMS ^posts ; The microwell array chip and the centrifuge tube were centrifuged at a speed of 600g for 1s; after the centrifugation, the chip was taken out and washed to remove excess fluorescent microspheres on the chip; see Figure 5 to calculate the single fluorescent microsphere capture rate of the chip Be 64.8 ± 12.6%, wherein, capture efficiency calculates according to formula (1):

Capture efficiency = number of microwells containing a single fluorescent microsphere/24*100% in each array. The centrifugal speed of 600g was replaced by 300g, and the capture rate of fluorescent microspheres was calculated to be 1.6±2.1%.

The centrifugation speed of 600g was replaced by 450g, and the capture rate of fluorescent microspheres was calculated to be 21.3±9.8%.

Example 5:

Put the chip provided in Example 1 into a sterile centrifuge tube containing PDMS posts; put 5 mL of single-cell suspension with a concentration of 3×10 ⁵ /mL, the nested addressable microwell array chip and the centrifuge tube Centrifuge at a speed of 600g and a centrifugation time of 1s; after the centrifugation, the chip is taken out and washed with PBS buffer to remove excess cells; the single cell capture rate of the chip is calculated to be 35.0±18.2%.

The survival rate of single cells was determined, and the cell viability was determined by fluorescein diacetate (FDA)/propidium iodide (PI) staining experiment, wherein the final concentration of FDA was 2 μg/mL, the final concentration of PI was 10 μg/mL, and the staining time was 30 minutes; Referring to Fig. 6, the calculated survival rate of captured single cells is 98.3±3.4%, indicating that the method is safe for cells.

For single cell culture, place the chip that captures single cells in a living cell workstation for culture, and conduct continuous observation; At 72h, 84h, and 96h, the microwells that initially captured single cells were tracked and photographed; see Figure 7, it can be observed that the cells are in normal shape and can proliferate normally, and at the same time, it is observed that single cells will gradually migrate out of the microwells that capture cells in the lower layer and enter the upper layer Cell culture microwells.

The descriptions of the above embodiments are only used to help understand the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection 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.

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