CN210765350U - Array micro-control chip for single cell capture and tumor ball culture - Google Patents
Array micro-control chip for single cell capture and tumor ball culture Download PDFInfo
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- CN210765350U CN210765350U CN201920811902.1U CN201920811902U CN210765350U CN 210765350 U CN210765350 U CN 210765350U CN 201920811902 U CN201920811902 U CN 201920811902U CN 210765350 U CN210765350 U CN 210765350U
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Abstract
The utility model discloses an arrayed micro-control chip that unicellular was caught and tumour ball was cultivateed, including unicellular sorting layer and cell culture layer, it has the cell culture layer to bond on the unicellular sorting layer, the unicellular sorting layer includes that at least a set of cell catches the array, cell suspension entry and cell suspension export, every group cell catches the array and includes at least three rows of microarray, every row of microarray is including arranging the unicellular unit of catching side by side, the unicellular unit of catching includes two microstructures, form the screening groove between two microstructures, the both ends of screening groove form respectively and are used for catching first hole and the second hole of cell, the width in first hole is 2 μm bigger than the width in second hole; the cell culture layer is provided with a culture groove, and the culture groove corresponds to the single cell capturing unit. The chip of the utility model has the characteristics of easy operation is swift, low consumption, can wide application in multiple parallel high flux and multiple double entry unicellular operation and analysis application.
Description
Technical Field
The utility model belongs to the technical field of cell biology and micro-fluidic chip, concretely relates to array micro-control chip that unicellular capture and tumour ball were cultivateed.
Background
Currently, single cell level-based research and analysis are widely used in biological research and clinical testing and are growing as an emerging analytical method and testing platform. The micro-fluidic chip technology is a micro-operation and analysis method which has just emerged in this century. The method shows extremely strong single cell operation and analysis potential at the beginning of creation. To date, the single cell capturing and operating method based on the microfluidic chip mainly includes: microstructural filtering, hydrodynamic manipulation, magnetic manipulation, optical manipulation, electric field manipulation, acoustic manipulation, and the like. The method for capturing and culturing the single cells for a long time based on the micro-structure filtering method is widely applied and developed due to the advantages of simple principle, quick operation, capability of developing real-time high flux, no need of assisting other instruments and the like. However, the current chips based on microstructure units (e.g., micro-dams and micro-wells) are difficult to study for long-term culture due to their limitations in their own principles. And the detection based on the long-term cell culture (such as stem cell culture, drug screening and the like) cannot be carried out. This not only wastes trapped cells, but also limits the scope of later experimental studies of trapped single cells to a large extent.
SUMMERY OF THE UTILITY MODEL
The utility model aims at providing a unicellular array micro-control chip of catching and tumour ball cultivation realizes single not equidimension and the unicellular catching of deformability through designing a complicated micro-filtration structure, and then realizes catching long-term cultivation and subsequent detection of unicellular through the combination with microporous structure, has solved the cell chip function singleness of current micro-filtration structure, can't carry out later stage cell culture and cause the extravagant problem of catching unicellular.
The utility model adopts the technical scheme that an arrayed micro-control chip for capturing single cells and culturing tumor balls comprises a single cell sorting layer, a cell culture layer and a pipeline embedded in the single cell sorting layer, wherein the single cell sorting layer is bonded with the cell culture layer, the single cell sorting layer comprises at least one group of cell capturing array, a cell suspension inlet and a plurality of cell suspension outlets, each group of cell capturing array is communicated with the cell suspension inlet and the plurality of cell suspension outlets through the pipeline, each group of cell capturing array comprises at least three rows of microarrays, each row of microarrays comprises a plurality of single cell capturing units arranged in parallel, each single cell capturing unit comprises two H-shaped microstructures, a screening groove used for screening the cells is formed between the two H-shaped microstructures, and the two ends of the screening groove respectively form a first pore and a second pore used for capturing the cells, the width of the first pore is 1-2 μm larger than that of the second pore;
a plurality of mutually independent culture tanks for culturing cells are uniformly arranged on the cell culture layer at intervals, the culture tanks are arranged in an array, and each culture tank corresponds to the single cell capturing unit one by one.
The utility model is also characterized in that,
preferably, the distance between two adjacent columns of microarrays is 50 μm.
Preferably, the cross section of each H-shaped microstructure is a rectangle with a radially contracted middle part, the length of each H-shaped microstructure is 200-300 μm, the width of each H-shaped microstructure at two ends of the rectangle is 120 μm, and the width of the contracted middle part is 90 μm.
Preferably, the screening slot is wide in the middle and narrow on both sides, the length of the screening slot is 100 to 150 μm, the width of the widest part in the middle is 40 to 60 μm, the height of the screening slot is the same as the height of the pipeline, and the height of the screening slot is 18 to 28 μm.
Preferably, the single-cell sorting layer is provided with a plurality of micro-columns at two sides of the cell capture array, and the distance between every two micro-columns is 25-50 μm for preventing the pipeline from collapsing.
Preferably, each culture vessel has a length of 80 to 120 μm, a width of 80 to 120 μm, and a height of 70 to 100 μm.
Preferably, the size of the first pores and the size of the second pores of the microarrays positioned in the same column are constant, the size of the first pores of the microarrays positioned in two adjacent columns is different by 2 μm, and the size of the second pores of the microarrays positioned in two adjacent columns is different by 2 μm.
The beneficial effects of the utility model are that, an array ization micro-control chip that unicellular was caught and tumour ball is cultivateed, through design unicellular sorting layer and cell culture layer, designed array structure's cell capture unit in unicellular sorting layer, designed array's culture tank in the culture layer, mutual independence between each culture tank realizes accomplishing unicellular catching, cultivation and subsequent detection operation in succession. Compared with the operation of capturing single cells by the micro structure in the traditional micro-fluidic chip, the micro-fluidic chip can capture and culture single cells with different sizes and deformability for a long time, and can be widely applied to various parallel high-throughput and multiple compound single cell operation and analysis applications.
Drawings
FIG. 1 is a schematic diagram of the overall structure of an arrayed micro-control chip for single cell capture and tumor ball culture according to the present invention;
FIG. 2 is a top view of a cell capture array in an arrayed micro-control chip for single cell capture and tumor ball culture according to the present invention;
FIG. 3 is a schematic cross-sectional view of a micro-structure in an arrayed micro-control chip for single-cell capture and tumor ball culture according to the present invention;
FIG. 4 is a schematic flow chart of the operation method for the representation of the micro-control chip side structure according to the present invention;
FIG. 5 is a schematic view showing that tumor cells having different sizes and deformability are poured into the micro-control chip in example 1; wherein, FIG. 5A is a white light map and a fluorescence map of the distribution of Normal U251 cells in the microstructure array, and FIG. 5B is a white light map and a fluorescence map of the distribution of Induced U251 cells in the microstructure array;
FIG. 6 is a statistical chart of the distribution of Normal U251 cells and Induced U251 cells with different sizes and deformability in the microstructure poured into the micro-control chip in example 1; wherein, FIG. 6A is a statistical chart of the percentage distribution of Normal U251 cells and Induced U251 cells in the microstructure; FIG. 6B is a statistical plot of the number of Normal U251 cells and Induced U251 cells in the microstructure; the microstructure size is shown as the size of the second pore of the single cell sorting layer cell-trapping microcell;
FIG. 7 white light map of tumor sphere formation by single cells in different microstructures of example 1; wherein the numbers above the figures represent the size in the pores between adjacent microstructures; FIG. 7A is a white light map of a Normal U251 cell; FIG. 7B white light map of InducedU251 cells; the microstructure size is shown as the size of the second pore of the single cell sorting layer cell-trapping microcell;
FIG. 8 shows the balling rates of single-cell derived tumor spheres in the microstructure of Normal U251 cells and Induced U251 cells in example 1;
FIG. 9 shows sizes of single-cell derived tumor spheres in the microstructure of Normal U251 cells and Induced U251 cells in example 1;
FIG. 10A is a graph of the balling rate of tumor sphere formation in microstructures for different size and deformability of individual tumors; the microstructure size is shown as the size of the second pore of the single cell sorting layer cell-trapping microcell;
FIG. 10B shows tumor sphere sizes for different sizes and deformability of individual tumors forming tumor spheres in the microstructure; the microstructure size is shown as the size of the second pore of the single cell sorting layer cell trapping microcell.
In the figure, 1, a single cell sorting layer, 2, a cell culture layer, 3, a pipeline, 4, a cell capture array, 5, a microarray, 6, a screening groove, 7, a single cell capture unit, 8, an H-shaped microstructure, 9, a culture groove, 10, a first pore, 11, and a second pore.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and specific embodiments.
The utility model discloses an arrayed micro-control chip of unicellular capture and tumor cell culture, as shown in figure 1, figure 2 and figure 3, including unicellular sorting layer 1, cell culture layer 2 and pipeline 3 of embedding in unicellular sorting layer 1, it has cell culture layer 2 to bond on unicellular sorting layer 1, unicellular sorting layer 1 includes that at least a set of cell catches array 4, a cell suspension entry and a plurality of cell suspension export, every group cell catches array 4 and communicates cell suspension entry and a plurality of cell suspension export through pipeline 3, every group cell catches array 4 and includes at least three rows of microarray 5, every row of microarray 5 includes a plurality of unicellular capture unit 7 of arranging side by side, every unicellular capture unit 7 includes two H type microstructures 8, form the screening groove 6 that is used for the cell screening between two H type microstructures 8, the both ends of screening groove 6 form first hole 10 and second hole 11 that are used for catching the cell respectively, the width of the first aperture 10 is 1 to 2 μm larger than that of the second aperture 11;
a plurality of mutually independent culture tanks 9 for culturing cells are uniformly arranged on the cell culture layer 2 at intervals, the culture tanks 9 are arranged in an array, and each culture tank 9 corresponds to the single cell capturing unit 7 one by one.
The pitch of two adjacent columns of microarrays 5 is 50 μm.
The cross section of each H-shaped microstructure 8 is a rectangle with a radially contracted middle part, the length of each H-shaped microstructure 8 is 200-300 mu m, the width of two ends of the rectangle of each H-shaped microstructure 8 is 120 mu m, and the width of the contracted middle part is 90 mu m.
The screening groove 6 is wide in the middle and narrow in two sides, the length of the screening groove 6 is 100-150 mu m, the width of the widest part in the middle is 40-60 mu m, the height of the screening groove 6 is the same as that of the pipeline 3, and the height of the screening groove 6 is 18-28 mu m.
The single cell separation layer 1 is provided with a plurality of micro-columns at two sides of the cell capture array, and the distance between every two micro-columns is 25-50 μm for preventing the pipeline from collapsing.
The sizes of the first pores 10 and the second pores 11 of the microarrays 5 in the same column are constant, the sizes of the first pores 10 of the microarrays 5 in two adjacent columns are different by 2 μm, and the sizes of the second pores 11 of the microarrays in two adjacent columns are different by 2 μm.
The length of each culture groove 9 is 80-120 mu m, the width is 80-120 mu m, the height is 70-100 mu m, the culture grooves 9 in the cell culture layer 2 are independent structures, and the culture grooves are not communicated with each other through pipelines, so that the independence of the captured single cells is ensured.
The utility model discloses a theory of operation of arrayed micro-control chip that unicellular capture and tumour ball were cultivateed as follows: the single cell capturing unit of the single cell sorting layer mainly captures single cells with different sizes and deformability, the culture tank cultures the captured single cells for a long time, and the array structure is favorable for capturing and culturing the single cells for a long time. The utility model discloses a cell damage is avoided to the structure of unicellular separation layer and unicellular separation efficiency is improved. Each micro-junction section in each group of cell capture array is a rectangle with a radially contracted middle part, two adjacent microstructures form a single cell capture unit, and the single cell capture unit is provided with two pores for cell capture: a first pore and a second pore, wherein the first pore is 2 μm wider than the second pore. The height of the channel of the whole single cell separation layer is 18-28 μm, and the size includes the size of most mammalian cells. The microstructure is in a shape of a rectangle with a radially contracted middle part, so that the flow velocity reaches peak values at two minimum cross sections (a first pore and a second pore) passing through the microstructure, and the flow velocity gradually decreases at the middle part of the culture tank, thereby providing a controllable fluid environment for capturing or passing cells, and avoiding potential blocking risks and cell damage caused by long-time contact of the cells and the capturing unit.
The single cell separation layer mainly captures single cells with different sizes and deformability, and the culture tank in the cell culture layer is used for culturing the captured single cells.
The utility model discloses the material on unicellular sorting layer and cell culture layer is PDMS polymer, through carrying out irreversible sealing-in with the PDMS polymer of different proportions, guarantees that the uniformity and the independence of little unit of little pipeline of single cell capture and cell culture in the chip.
The utility model discloses a preparation method of arrayed micro-control chip that unicellular is caught and tumour ball cultivates specifically includes following step:
mixing a PDMS matrix and a curing agent according to a mass ratio of 5:1, simultaneously treating a single cell sorting layer mould by using Trimethylchlorosilane (TMCS) steam for 5-10 min, pouring the mixture of the PDMS matrix and the curing agent onto the single cell sorting layer mould treated by the trimethylchlorosilane, vacuumizing, degassing, heating and curing for 0.5-1 h in an oven at 80-100 ℃, stripping the cured PDMS from the mould, cutting according to requirements, punching to prepare a screening groove, and cleaning for later use, wherein the curing agent is purchased from Dow Corning company of America and is numbered SYLGARD 184.
mixing a PDMS matrix and a curing agent according to a mass ratio of 15:1, treating a cell culture layer mould for 5min by using TMCS steam, pouring the PDMS matrix and the curing agent into the cell culture layer mould treated by TMCS, vacuumizing and degassing the mould in an oven at the temperature of 80-100 ℃, heating and curing the mould for 1-2 h, stripping the cured PDMS from the mould, cutting the PDMS according to the requirement, punching the mould to prepare a culture tank, and cleaning the culture tank for later use;
the alignment error of the micro-pore chip can not exceed 10 mu m, so that the alignment is carried out by adopting an inverted fluorescence microscope with higher precision, specifically, a single cell sorting layer or a cell culture layer is placed in the visual field of the inverted fluorescence microscope, then the other layer is fixed by using a transparent adhesive tape after being aligned and is placed in an oven with the temperature of 80-100 ℃, a micro-control chip is obtained after heating and bonding for 50-100 h, the micro-control chip is taken out, the transparent adhesive tape is taken off, and the gap along the side surface of the micro-control chip is coated with glue to seal the micro-control chip for later use.
And 3, sealing the gap on the side surface of the micro-control chip, namely mixing the PDMS matrix and the curing agent according to the mass ratio of 8-15: 1, smearing the mixture on the gap on the side surface of the micro-control chip, placing the mixture in an oven at the temperature of 80-100 ℃, and heating and bonding for 50-100 hours to firmly bond the mixture.
The utility model discloses an operating method of array micro-control chip that unicellular is caught and tumour ball cultivates, as shown in figure 4, concrete operation includes following step:
Under a certain flow velocity, cell suspension is poured into a plurality of microstructures from a cell suspension inlet of a single cell separation layer, the cell suspension passes through the microstructures, large cells or cells with poor deformability pass through first pores and are clamped and captured by second pores, and small cells or cells with strong deformability pass through the first pores and the second pores and are not captured, so that the small cells or the cells with the poor deformability pass through a screening groove of the microstructure with smaller pores behind;
and 2, stably distributing the cells to be captured in the capturing units, turning over the micro-control chip, standing to enable the captured cells to enter a culture tank of the cell culture layer from the single cell capturing layer under the action of gravity, and then perfusing cell culture solution, clinical drugs and detection reagents through a cell suspension inlet at a slow flow rate to perform cell culture.
Example 1
The structure and size of the micro-control chip designed in the applicant's laboratory, as shown in fig. 2, a and B respectively represent the width and length of the micro-structure in the single cell sorting layer, and respectively take values of 120 μm and 200 μm, the width of the middle constriction is 90 μm, C represents the distance between the micro-arrays and takes values of 50 μm, D and E represent the same size in the same row of capturing units for the first pore and the second pore between two adjacent micro-structures, but the size is reduced by 2 μm in different capturing units for the micro-arrays in sequence (i.e. the size of the first pore is 16 to 8 μm and the size of the second pore is 14 to 6 μm in this embodiment), as shown in fig. 3, the length of the screening groove formed between two adjacent micro-structures is 100 μm, the width of the widest position in the middle is 40 μm, the height L1 of the screening groove of the single cell sorting layer 1 is 18 μm, the length and width of the culture grooves of the cell culture layer 2 are all 80 μm, the height L2 is 70 μm, and the culture grooves correspond to the microstructures of the single cell separation layer one by one.
The preparation process of the micro-control chip is as follows:
mixing a PDMS matrix and a curing agent according to a mass ratio of 5:1, simultaneously treating a single cell sorting layer mould with Trimethylchlorosilane (TMCS) steam for 5min, pouring the mixture of the PDMS matrix and the curing agent onto the single cell sorting layer mould treated by the trimethylchlorosilane, vacuumizing, degassing, heating and curing in an oven at 80 ℃ for 0.5h, peeling the cured PDMS from the mould, cutting, perforating, and cleaning for later use, wherein the PDMS matrix and the curing agent are purchased from Dow Corning company of America and have the number of SYLGARD 184.
mixing a PDMS matrix and a curing agent according to a mass ratio of 15:1, treating the cell culture layer mold with TMCS steam for 5min, pouring the PDMS matrix and the curing agent into the cell culture layer mold treated by TMCS, vacuumizing and degassing the cell culture layer mold in an oven at 80 ℃, heating and curing for 1h, peeling the cured PDMS from the mold, cutting, and cleaning for later use;
and 3, placing the single cell separation layer or the cell culture layer in the visual field of an inverted fluorescence microscope, then fixing the other layer after aligning by using a transparent adhesive tape, placing the fixed layer in an oven at 80 ℃, heating and bonding for 50h to obtain the micro-control chip, taking out the transparent adhesive tape, and smearing glue along the gap on the side surface of the micro-control chip to seal the micro-control chip.
An operation method of a single cell capture and tumor sphere culture arrayed micro-control chip cultured cells is shown in fig. 4, the tumor cells adopted are human glioma cells (U251) obtained from shanghai research institute of chinese academy of sciences, two different tumor cells (Normal U251 cells and Induced U251 cells) are common U251(Normal U251 cells) cultured by a method of DMEM/F12+ 10% FBS (fetal bovine serum), and Induced U251 cells (Induced U251) cultured by DMEM/F12 added with B27(1 ×), recombinant human epidermal growth factor (20ng/mL), basic fibroblast growth factor (20ng/mL) and leukemia inhibitory factor (10 ng/mL);
under the conditions that the driving flow rate is 40 mul/min and the cell density in cell samples (Normal U251 cells and Induced U251 cells) is 10000cells/mL, pouring the cell samples into a micro-control chip from a cell suspension inlet of a single cell separation layer for 20s, and respectively pouring the cell samples into a plurality of microstructures through uniformly distributed pipelines; the micro-structure array is passed at 100. mu.l/min so that the cell sample can be captured and separated by the micro-structure array according to different sizes and deformability. Cells with large or poor deformability can be clamped by the rear end gap through the front end gap, and cells with small or strong deformability can pass through the two gaps without being captured, so that the cells can be arranged in a microstructure array with smaller gaps behind; then, the cells to be captured are stably distributed in the microstructure array, the micro-control chip is turned over and stands still, so that the captured cells enter the micro-culture groove of the cell culture layer from the single cell capture layer through the action of gravity to form a tumor sphere, and then cell culture solution, clinical drugs, detection reagents and the like are perfused through an inlet at a slow flow rate of 10 mu l/min.
As shown in FIG. 5, the distribution of two different sizes and deformed tumor cells (Normal U251 cells and Induced U251 cells) in the microstructure array after perfusion at 100. mu.l/min with a cell concentration of 10000cells/mL in the cell sample is clearly different.
As shown in FIG. 6, the distribution of two different sizes and deformed tumor cells (Normal U251 cells and Induced U251 cells) in the microstructure array after perfusion at 100. mu.l/min with a cell concentration of 10000cells/mL in the cell sample is clearly different from that of Normal U251 cells (A) and Induced U251 cells (B).
After the tumor cells different from Normal U251 cells (A) and Induced U251 cells (B) were distributed and stabilized in the microstructure array, the cells were perfused with cell culture medium (DMEM/F12+ FBS 10%) at 10. mu.l/min, and observed and photographed for a certain period of time (0 days, 5 days, and 10 days), as shown in FIG. 7. From the figure, it can be seen that as the culture time increases, the single tumor cells gradually proliferate in the micro-culture tank to form tumor spheres, and almost all of the tumor spheres are formed by the culture until the 10 th day.
As shown in FIGS. 8 and 9, it can be seen from the results that the tumorigenicity rate of the Induced U251 cells was higher than that of the NormalU251 cells; it was seen that the tumor spheres formed by a single Induced U251 cell were larger than that of a single Normal U251 cell.
As shown in FIG. 10, the comparison showed that the spheroidisation rate and the size of the formed tumor spheres were higher for the small cells and/or the cells with large deformability than for the large cells and/or the cells with weak deformability.
Example 2
The structure and size of the micro-control chip designed in the applicant's laboratory, as shown in fig. 2, a and B respectively represent the width and length of the micro-structure in the single cell sorting layer, and respectively take the values of 120 μm and 250 μm, the width of the middle constriction is 90 μm, C represents the distance between the micro-arrays and takes the values of 50 μm, D and E represent that the first pore and the second pore between two adjacent micro-structures are the same in the same column of capturing units, but the sizes thereof are sequentially reduced by 2 μm in the capturing units of different micro-arrays (i.e. the size of the first pore is 16 to 8 μm and the size of the second pore is 14 to 6 μm in this embodiment), as shown in fig. 3, the length of the screening groove formed between two adjacent micro-structures is 120 μm, the width of the widest part in the middle is 50 μm, the height L1 of the screening groove of the single cell sorting layer 1 is 25 μm, the culture grooves of the cell culture layer 2 are all 100 μm in length and width and 75 μm in height L2, and correspond to the microstructures of the single cell separation layer one by one.
mixing a PDMS matrix and a curing agent according to a mass ratio of 5:1, simultaneously treating a single-cell sorting layer mold for 7min by using Trimethylchlorosilane (TMCS) steam, pouring the mixture of the PDMS matrix and the curing agent onto the single-cell sorting layer mold treated by the trimethylchlorosilane, vacuumizing, degassing, placing in an oven at 90 ℃ for heating and curing for 0.7h, peeling the cured PDMS from the mold, cutting, perforating, and cleaning for later use, wherein the curing agent is purchased from Dow Corning company of America, and has a serial number of SYLGARD 184.
mixing a PDMS matrix and a curing agent according to a mass ratio of 15:1, treating the cell culture layer mold with TMCS steam for 5min, pouring the PDMS matrix and the curing agent into the cell culture layer mold treated by TMCS, vacuumizing and degassing the cell culture layer mold in a drying oven at 90 ℃, heating and curing for 1.5h, peeling the cured PDMS from the mold, cutting, and punching and cleaning the mold for later use;
and 3, placing the single cell separation layer or the cell culture layer in the visual field of an inverted fluorescence microscope, then fixing the other layer by using a transparent adhesive tape after aligning, placing the other layer in a drying oven at 90 ℃, heating and bonding for 80 hours to obtain the micro-control chip, taking out the transparent adhesive tape, and smearing glue along the gap on the side surface of the micro-control chip to seal the micro-control chip.
Example 3
The structure and size of the micro-control chip designed in the applicant's laboratory, as shown in fig. 2, a and B respectively represent the width and length of the micro-structure in the single-cell separation layer, and respectively take values of 120 μm and 300 μm, the width of the middle constriction is 90 μm, C represents the distance between the micro-arrays and takes values of 50 μm, D and E represent the first pore and the second pore between two adjacent micro-structures are the same in the same column of capture units, but the size is reduced by 2 μm in different capture units of the micro-arrays in sequence (i.e. the size of the first pore is 16 to 8 μm in this embodiment, the size of the second pore is 14 to 6 μm), as shown in fig. 3, the length of the screening groove formed between two adjacent micro-structures is 150 μm, the width of the widest position in the middle is 60 μm, the height L1 of the screening grooves of the single cell separation layer 1 is 28 μm, the length and width of the culture grooves of the cell culture layer 2 are 120 μm, the height L2 is 100 μm, and the culture grooves correspond to the microstructures of the single cell separation layer one by one.
mixing a PDMS matrix and a curing agent according to a mass ratio of 5:1, simultaneously treating a single cell sorting layer mould with Trimethylchlorosilane (TMCS) steam for 10min, pouring the mixture of the PDMS matrix and the curing agent onto the single cell sorting layer mould treated by the trimethylchlorosilane, vacuumizing, degassing, heating and curing in an oven at 100 ℃ for 1h, peeling the cured PDMS from the mould, cutting, perforating, and cleaning for later use, wherein the PDMS matrix and the curing agent are purchased from Dow Corning company of America and have the serial number of SYLGARD 184.
mixing a PDMS matrix and a curing agent according to a mass ratio of 15:1, treating the cell culture layer mold with TMCS steam for 5min, pouring the PDMS matrix and the curing agent into the cell culture layer mold treated by TMCS, vacuumizing and degassing the cell culture layer mold in an oven at 100 ℃, heating and curing for 2h, peeling the cured PDMS from the mold, cutting, and cleaning the mold for later use;
and 3, placing the single cell separation layer or the cell culture layer in the visual field of an inverted fluorescence microscope, then fixing the other layer by using a transparent adhesive tape after aligning, placing the other layer in a drying oven at 100 ℃, heating and bonding for 100 hours to obtain the micro-control chip, taking out the transparent adhesive tape, and smearing glue along the gap on the side surface of the micro-control chip to seal the micro-control chip.
Example 4
The structure and size of the micro-control chip designed in the applicant's laboratory, as shown in fig. 2, a and B respectively represent the width and length of the micro-structure in the single-cell separation layer, and respectively take values of 120 μm and 300 μm, the width of the middle constriction is 90 μm, C represents the distance between the micro-arrays and takes values of 50 μm, D and E represent the first pore and the second pore between two adjacent micro-structures are the same in the same column of capture units, but the size is reduced by 2 μm in different capture units of the micro-arrays in sequence (i.e. the size of the first pore is 16 to 8 μm in this embodiment, the size of the second pore is 14 to 6 μm), as shown in fig. 3, the length of the screening groove formed between two adjacent micro-structures is 150 μm, the width of the widest position in the middle is 60 μm, the height L1 of the screening grooves of the single cell separation layer 1 is 28 μm, the length and width of the culture grooves of the cell culture layer 2 are 120 μm, the height L2 is 100 μm, and the culture grooves correspond to the microstructures of the single cell separation layer one by one.
mixing a PDMS matrix and a curing agent according to a mass ratio of 5:1, simultaneously treating a single cell sorting layer mould with Trimethylchlorosilane (TMCS) steam for 10min, pouring the mixture of the PDMS matrix and the curing agent onto the single cell sorting layer mould treated by the trimethylchlorosilane, vacuumizing, degassing, heating and curing in an oven at 100 ℃ for 1h, peeling the cured PDMS from the mould, cutting, perforating, and cleaning for later use, wherein the PDMS matrix and the curing agent are purchased from Dow Corning company of America and have the serial number of SYLGARD 184.
mixing a PDMS matrix and a curing agent according to a mass ratio of 15:1, treating the cell culture layer mold with TMCS steam for 5min, pouring the PDMS matrix and the curing agent into the cell culture layer mold treated by TMCS, vacuumizing and degassing the cell culture layer mold in an oven at 100 ℃, heating and curing for 2h, peeling the cured PDMS from the mold, cutting, and cleaning the mold for later use;
and 3, placing the single cell separation layer or the cell culture layer in the visual field of an inverted fluorescence microscope, then aligning the other layer, fixing the other layer by using a transparent adhesive tape, placing the other layer in a 100 ℃ oven, heating and bonding for 100 hours to obtain a micro-control chip, taking out the micro-control chip, removing the transparent adhesive tape, mixing the PDMS matrix and a curing agent according to the mass ratio of 8:1, coating the mixture at a gap on the side surface of the micro-control chip, placing the micro-control chip in the 80 ℃ oven, and heating and bonding for 50 hours to firmly bond the PDMS matrix and the curing agent.
Example 5
The structure and size of the micro-control chip designed in the applicant's laboratory, as shown in fig. 2, a and B respectively represent the width and length of the micro-structure in the single-cell separation layer, and respectively take the values of 120 μm and 200 μm, the width of the middle constriction is 90 μm, C represents the distance between the micro-arrays and takes the value of 50 μm, D and E represent the first pore and the second pore between two adjacent micro-structures are the same in the same row of capture units, but the size is reduced by 2 μm in different capture units of the micro-arrays in sequence (i.e. the size of the first pore is 16 to 8 μm in this embodiment, the size of the second pore is 14 to 6 μm), as shown in fig. 3, the length of the screening groove formed between two adjacent micro-structures is 150 μm, the width of the widest part in the middle is 60 μm, the height L1 of the screening grooves of the single cell separation layer 1 is 25 μm, the length and width of the culture grooves of the cell culture layer 2 are 120 μm, the height L2 is 75 μm, and the culture grooves correspond to the microstructures of the single cell separation layer one by one.
mixing a PDMS matrix and a curing agent according to a mass ratio of 5:1, simultaneously treating a single cell sorting layer mould with Trimethylchlorosilane (TMCS) steam for 10min, pouring the mixture of the PDMS matrix and the curing agent onto the single cell sorting layer mould treated by the trimethylchlorosilane, vacuumizing, degassing, heating and curing in an oven at 100 ℃ for 1h, peeling the cured PDMS from the mould, cutting, perforating, and cleaning for later use, wherein the PDMS matrix and the curing agent are purchased from Dow Corning company of America and have the serial number of SYLGARD 184.
mixing a PDMS matrix and a curing agent according to a mass ratio of 15:1, treating the cell culture layer mold with TMCS steam for 5min, pouring the PDMS matrix and the curing agent into the cell culture layer mold treated by TMCS, vacuumizing and degassing the cell culture layer mold in an oven at 100 ℃, heating and curing for 2h, peeling the cured PDMS from the mold, cutting, and cleaning the mold for later use;
and 3, placing the single cell separation layer or the cell culture layer in the visual field of an inverted fluorescence microscope, then aligning the other layer, fixing the other layer by using a transparent adhesive tape, placing the other layer in a 100 ℃ oven, heating and bonding for 100 hours to obtain a micro-control chip, taking out the micro-control chip, removing the transparent adhesive tape, mixing the PDMS matrix and a curing agent according to the mass ratio of 10:1, coating the mixture at a gap on the side surface of the micro-control chip, placing the micro-control chip in the 90 ℃ oven, and heating and bonding for 100 hours to firmly bond the PDMS matrix and the curing agent.
Example 6
The structure and size of the micro-control chip designed in the applicant's laboratory, as shown in fig. 2, a and B respectively represent the width and length of the micro-structure in the single-cell separation layer, and respectively take the values of 120 μm and 200 μm, the width of the middle constriction is 90 μm, C represents the distance between the micro-arrays and takes the value of 50 μm, D and E represent the first pore and the second pore between two adjacent micro-structures are the same in the same column of capture units, but the size is reduced by 2 μm in different capture units of the micro-arrays in sequence (i.e. the size of the first pore is 16 to 8 μm in this embodiment, the size of the second pore is 14 to 6 μm), as shown in fig. 3, the length of the screening groove formed between two adjacent micro-structures is 120 μm, the width of the widest position in the middle is 50 μm, the height L1 of the screening grooves of the single cell separation layer 1 is 28 μm, the length and width of the culture grooves of the cell culture layer 2 are 100 μm, the height L2 is 100 μm, and the culture grooves correspond to the microstructures of the single cell separation layer one by one.
mixing a PDMS matrix and a curing agent according to a mass ratio of 5:1, simultaneously treating a single cell sorting layer mould with Trimethylchlorosilane (TMCS) steam for 10min, pouring the mixture of the PDMS matrix and the curing agent onto the single cell sorting layer mould treated by the trimethylchlorosilane, vacuumizing, degassing, heating and curing in an oven at 100 ℃ for 1h, peeling the cured PDMS from the mould, cutting, perforating, and cleaning for later use, wherein the PDMS matrix and the curing agent are purchased from Dow Corning company of America and have the serial number of SYLGARD 184.
mixing a PDMS matrix and a curing agent according to a mass ratio of 15:1, treating the cell culture layer mold with TMCS steam for 5min, pouring the PDMS matrix and the curing agent into the cell culture layer mold treated by TMCS, vacuumizing and degassing the cell culture layer mold in an oven at 100 ℃, heating and curing for 2h, peeling the cured PDMS from the mold, cutting, and cleaning the mold for later use;
and 3, placing the single cell separation layer or the cell culture layer in the visual field of an inverted fluorescence microscope, then aligning the other layer, fixing the other layer by using a transparent adhesive tape, placing the other layer in a 100 ℃ oven, heating and bonding for 100 hours to obtain a micro-control chip, taking out the micro-control chip, removing the transparent adhesive tape, mixing the PDMS matrix and a curing agent according to the mass ratio of 15:1, coating the mixture at a gap on the side surface of the micro-control chip, placing the micro-control chip in the 100 ℃ oven, and heating and bonding for 85 hours to firmly bond the PDMS matrix and the curing agent.
Claims (7)
1. An arrayed micro-control chip for single cell capture and tumor cell culture, which comprises a single cell sorting layer (1), a cell culture layer (2) and a pipeline (3) embedded in the single cell sorting layer (1), wherein the single cell sorting layer (1) is adhered with the cell culture layer (2), the single cell sorting layer comprises at least one group of cell capture arrays (4), a cell suspension inlet and a plurality of cell suspension outlets, each group of cell capture arrays (4) is communicated with the cell suspension inlet and the plurality of cell suspension outlets through the pipeline (3), each group of cell capture arrays (4) comprises at least three rows of microarrays (5), each row of microarrays (5) comprises a plurality of single cell capture units (7) arranged in parallel, each single cell capture unit (7) comprises two H-shaped microstructures (8), a screening groove (6) for cell screening is formed between the two H-shaped microstructures (8), a first pore (10) and a second pore (11) for capturing cells are respectively formed at two ends of the screening groove (6), and the width of the first pore (10) is 1-2 mu m larger than that of the second pore (11);
a plurality of mutually independent culture tanks (9) for culturing cells are uniformly arranged on the cell culture layer (2) at intervals, the culture tanks (9) are arranged in an array, and each culture tank (9) corresponds to the single-cell capturing unit (7) one by one.
2. The arrayed micro-control chip for single-cell capture and tumor sphere culture of claim 1, wherein the distance between two adjacent columns of the micro-arrays (5) is 50 μm.
3. The arrayed micro-control chip for single-cell capture and tumor sphere culture according to claim 1, wherein each H-shaped microstructure (8) has a cross section in a rectangular shape with a radially contracted middle part, each H-shaped microstructure (8) has a length of 200 μm to 300 μm, the width of each rectangular H-shaped microstructure is 120 μm at two ends, and the width of the contracted middle part is 90 μm.
4. The arrayed micro-control chip for single-cell capture and tumor sphere culture according to claim 1, wherein the screening slot (6) is wide in the middle and narrow in both sides, the length of the screening slot (6) is 100 μm to 150 μm, the width of the widest part in the middle is 40 μm to 60 μm, the height of the screening slot (6) is the same as the height of the pipeline (3), and the height of the screening slot (6) is 18 μm to 28 μm.
5. The arrayed micro-control chip for single-cell capture and tumor sphere culture according to claim 1, wherein the single-cell separation layer (1) is provided with a plurality of micro-columns at two sides of the cell capture array, and the distance between each micro-column is 25 μm to 50 μm for preventing the pipeline (3) from collapsing.
6. The arrayed micro-control chip for single cell capture and tumor sphere culture according to claim 1, wherein each culture groove (9) has a length of 80 μm to 120 μm, a width of 80 μm to 120 μm, and a height of 70 μm to 100 μm.
7. The arrayed micro-control chip for single-cell capture and tumor sphere culture of claim 1, wherein the sizes of the first pores (10) and the second pores (11) of the microarrays (5) in the same column are constant, the sizes of the first pores (10) of the microarrays (5) in two adjacent columns are different by 2 μm, and the sizes of the second pores (11) of the microarrays in two adjacent columns are different by 2 μm.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110305788A (en) * | 2019-05-31 | 2019-10-08 | 西安医学院 | The array chip and its preparation and operating method of cell capture and the culture of tumour ball |
| CN111774110A (en) * | 2020-06-28 | 2020-10-16 | 中国科学院大连化学物理研究所 | Biological analysis chip capable of realizing cell capture and fixation |
| CN113088447A (en) * | 2021-03-11 | 2021-07-09 | 温州医科大学 | Suspended array microfluidic chip and preparation method and application thereof |
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2019
- 2019-05-31 CN CN201920811902.1U patent/CN210765350U/en active Active
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110305788A (en) * | 2019-05-31 | 2019-10-08 | 西安医学院 | The array chip and its preparation and operating method of cell capture and the culture of tumour ball |
| CN111774110A (en) * | 2020-06-28 | 2020-10-16 | 中国科学院大连化学物理研究所 | Biological analysis chip capable of realizing cell capture and fixation |
| CN113088447A (en) * | 2021-03-11 | 2021-07-09 | 温州医科大学 | Suspended array microfluidic chip and preparation method and application thereof |
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