CN114058502A - Biological analysis chip capable of realizing three-dimensional co-culture and multi-index detection of cells - Google Patents
Biological analysis chip capable of realizing three-dimensional co-culture and multi-index detection of cells Download PDFInfo
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N2333/54—Interleukins [IL]
- G01N2333/5412—IL-6
Abstract
The invention relates to the fields of microfluidic chips, bioanalysis and application, in particular to a bioanalysis chip capable of realizing three-dimensional co-culture and multi-index detection of cells. The micro-pore array chip comprises a biological analysis substrate and a three-dimensional micro-column substrate, and comprises a micro-column array substrate and a micro-pore array perforated membrane. The chip can realize high-flux cell three-dimensional co-culture, and various cells are distributed in the micropores differently to provide different three-dimensional physiological microenvironments for the cells. The micro-pore array chip is covered with a biological analysis substrate to realize multi-index biological analysis of three-dimensional co-culture of cells. The chip of the invention can be used for multi-index biological analysis of single cell three-dimensional culture and multiple cell three-dimensional co-culture, including protein secretion.
Description
Technical Field
The invention relates to the fields of microfluidic chips, bioanalysis and application, in particular to a bioanalysis chip capable of realizing three-dimensional co-culture and multi-index detection of cells.
Background
One key limitation of single cell analysis is that the analyzed cells are separated from the original microenvironment, and the analysis result cannot completely reflect the real state of the cells. The cell microenvironment is constructed in the single cell analysis, and the application of the single cell analysis in biology, immunology research, clinical diagnosis and evaluation can be expanded. Cellular microenvironments include physical microenvironments and physiological microenvironments. The morphology of the substrate in the physical microenvironment influences the morphology and function of the cells. Compared with a two-dimensional substrate, the state and the function of the cells cultured on the three-dimensional substrate can reflect the real situation in vivo. Therefore, three-dimensional culture provides a better culture mode for understanding the physiological and pathological mechanisms of cells. The existing single cell analysis technology is based on a two-dimensional culture mode, and the development of three-dimensional substrate single cell analysis is limited due to the technical bottleneck.
The physiological microenvironment of cells is another important factor affecting cell function. The in vitro simulated physiological microenvironment mainly adopts one or more cell co-culture modes to construct intercellular communication and matrix microenvironment. The cell physiological microenvironment is integrated in the single cell analysis, so that the interaction and mechanism between cells can be understood more deeply, and the development and application of the single cell analysis technology are an important supplement, but the current report is rare.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a multi-index biological analysis chip capable of realizing three-dimensional co-culture of cells, which can realize single cell and multi-cell analysis in a cell co-culture mode on a three-dimensional substrate and provide a new technology for in vitro deep understanding of cell mechanisms, functions and intercellular interaction and expanding single cell analysis and application.
In order to realize the purpose, the invention adopts the following technical scheme:
the invention provides a micro-pore array chip of a three-dimensional micro-column substrate, which comprises a substrate and a micro-pore array perforated membrane, wherein the substrate is a micro-column array substrate.
In the above technical solution, further, the cross section of the microcolumn is circular, regular polygon or rectangle, and the area of the cross section of the microcolumn is 70 × 10-6μm2-70μm2The height of the microcolumns is 100nm-1cm, and the distance between the boundaries of two adjacent microcolumns is 100nm-1 cm.
In the above technical solution, further, the microporous array perforated membrane is composed of 1 or more microporous arrays, each of the microporous arrays is in the shape of a regular polygon, a rectangle or a circle, the shape of the micropores includes a circle, a regular polygon or a rectangle, and the area of the cross section of the micropore is 0.5 μm2-2×105μm2The distance between the boundaries of two adjacent micropores is 100 mu m-1 mm; the thickness of the micropore array perforated film is 1 mu m-1 mm.
In the above technical solution, the substrate and the microporous array perforated membrane are made of one or more composite materials selected from PDMS, PMMA, PC, PS, POE, EVA, and EPDM, or one or more composite materials selected from PDMS, PMMA, PC, PS, POE, EVA, and EPDM after hydrophilic modification.
The invention provides a biological analysis chip capable of realizing three-dimensional co-culture of cells, which comprises a biological analysis substrate and the micropore array chip, wherein the biological analysis substrate is connected with the micropore array perforated membrane.
In the above technical solution, the material of the biological analysis substrate is polylysine, epoxy resin, amino group, aldehyde group modified glass or epoxy resin glass slide.
In the above technical solution, the glass or epoxy resin slide further includes a coated antibody, a protein or DNA, or a coated protein array, an antibody array, or a DNA array.
The third aspect of the invention provides the application of the micro-pore array chip of the three-dimensional micro-column substrate, which is applied to the three-dimensional culture or co-culture of one or more single cells; the chip is applied to three-dimensional co-culture of one or more kinds of multiple cells.
The fourth aspect of the invention provides an application of the micro-pore array chip with the three-dimensional micro-column substrate, wherein the application is single-cell and multi-cell three-dimensional co-culture analysis of various cells, single-cell multi-biomolecule analysis of various cells and multi-biomolecule analysis of various cells.
In the above technical solution, further, the multiple biomolecule analysis includes analysis of secreted proteins, cytokines, growth factors, cell surface proteins, cell surface receptors, polypeptides, nucleic acids, hormones, antigens or exosomes, and the multiple analysis includes analysis of 1 to 100 indices.
Compared with the prior art, the invention has the beneficial effects that:
the invention constructs a cell three-dimensional co-culture multi-index biological analysis platform, takes a three-dimensional micro-column array as a substrate, and integrates a microporous array perforated membrane on the three-dimensional micro-column array substrate to form a microporous array chip of the three-dimensional micro-column substrate, wherein the chip is a detachable and reusable chip; the substrate provides a three-dimensional substrate physical microenvironment for cell culture, the perforated membrane provides a micro-chamber space for three-dimensional culture of cells in the hole, and the perforated membrane is simultaneously used as a connecting layer to connect an upper biological micro-column array analysis substrate.
The micro-pore array chip with the three-dimensional micro-column substrate can be applied to single-cell and multi-cell three-dimensional co-culture of various cells and three-dimensional co-culture of various cells in different proportions, and provides a plurality of physiological micro-environments for the three-dimensional co-culture of cells.
The multi-index biomolecule analysis of the three-dimensional co-culture of the cells can broaden the research of single cell analysis in biology and medicine, and provides important technical support for disease diagnosis, treatment monitoring and evaluation application.
Drawings
FIG. 1 is a schematic diagram of a micro-pore array chip structure of a three-dimensional micro-column array substrate according to the present invention;
FIG. 2 is a schematic top view of a micro-well array chip with a three-dimensional micro-column array substrate according to the present invention;
FIG. 3 is a schematic diagram of the chip structure of the chip of the present invention when applied to the detection of multi-index secreted protein;
FIG. 4 is a schematic view of a method for fabricating a micro-well array chip of a three-dimensional micro-column array substrate according to the present invention;
FIG. 5 is a microscopic picture of a three-dimensional microcolumn array according to the present invention; a is a 10-fold bright field map, B is a 40-fold bright field map, and a scale shows 50 μm;
FIG. 6 is a scanning electron microscope image of a micro-pore array chip of the three-dimensional micro-column array substrate according to the present invention; a is a micropore array chip electron microscope picture taking a three-dimensional microcolumn array as a substrate, B is a chip electron microscope enlarged view in a mark frame in the picture A, and C is a chip electron microscope enlarged view in a mark frame in the picture B;
FIG. 7 is a graph of morphological analysis of cell culture applied in example 2; a is the form of MCF-7 cells on a PDMS substrate (P-PDMS) without a micro-column structure, and B is the form of MCF-7 cells on a micro-column array substrate (M-pilar) of the invention; c is a cell spreading area statistical result scatter diagram of MCF-7 on the micro-column-free substrate and the micro-column substrate of the invention, and D is a roundness statistical result scatter diagram of MCF-7 on the micro-column-free substrate and the micro-column substrate of the invention; scale bar represents 100 μm;
FIG. 8 is a macro-scanning and magnified microscopy brightfield and fluorescence image applied to three-dimensional co-culture of U937 macrophages and tumor cells in example 3; a is a co-cultured large image scanning bright field image, B is a bright field enlarged image in a mark frame of the image A, C is a single U937 macrophage fluorescence image in a micropore, D is a fluorescence image of a plurality of MCF-7 cells in the micropore, and a ruler shows 100 mu m;
FIG. 9 shows the cell distribution statistics applied to three-dimensional co-culture of cells in example 3; a is the distribution of U937 macrophages within the microwells and B is the distribution of MCF-7 tumor cell numbers in microwells for single U937 macrophages;
FIG. 10 shows the statistics of cell activities applied to three-dimensional co-culture of cells in example 3;
FIG. 11 shows the results of the cellular multi-index secreted protein assay and statistics applied in example 4; a is a TNF-a secretion rate change diagram of MCF-7 tumor co-culture or MCF-7 tumor co-culture on a three-dimensional micro-column array substrate and a micro-pore array PDMS chip without the micro-column substrate, and B is an IL-6 secretion rate change diagram of MCF-7 tumor co-culture or MCF-7 tumor co-culture on the three-dimensional micro-column array substrate and the micro-pore array PDMS chip without the micro-column substrate;
FIG. 12 shows the effect of different tumor microenvironments on protein secretion from U937 macrophages, applied to three-dimensional co-culture of cells in example 4; a is a graph of the change in the secretion rate of TNF-a from a single U937 macrophage cell line with increasing MCF-7 cell line, and B is a graph of the change in the secretion rate of IL-6 from a single U937 macrophage cell line with increasing MCF-7 cell line;
FIG. 13 is a perspective view of a chip for bioassay according to the present invention;
in the figure, 1, a biological analysis substrate, 2, a micropore array chip of a three-dimensional micro-column array substrate, 3, a substrate, 4, a micropore array perforated membrane, 5, a clamp, 6, a three-dimensional micro-column array silicon template, 7, a micropore array perforated membrane silicon template, and 8 are micropores of the micropore array perforated membrane.
Detailed Description
The invention is further illustrated but is not in any way limited by the following specific examples.
Example 1
A multi-index biological analysis chip for cell co-culture on a three-dimensional substrate is shown in figures 1-4. The chip is divided into three layers, including a biological analysis substrate 1, a three-dimensional micro-column array substrate 3 and a micropore array perforated membrane 4 from top to bottom. The micropore array perforated film 4 and the three-dimensional micro-column array substrate 3 form a micropore array chip 2 of the three-dimensional micro-column substrate, and the micropore array perforated film 4 and the three-dimensional micro-column array substrate 3 are aligned and tiled to obtain the three-dimensional micropore array chip 2 taking the three-dimensional micro-column array as the substrate. The cavity formed by the micropores 8 of the micropore array perforated membrane and the substrate 3 provides a microchamber space for three-dimensional cell culture, and the micropore array perforated membrane is connected with an upper layer biological analysis substrate.
Preferably, the micro-pore array chip 2 of the three-dimensional micro-column substrate is a micro-pore array chip of a PDMS three-dimensional micro-column substrate.
The micro-pore array chip of the three-dimensional micro-column array substrate is manufactured by the MEMS technology and the soft lithography method, and as shown in figure 4, the method specifically comprises the following steps:
mixing PDMS precursor A glue and cross-linking agent B glue according to a mass ratio of 10:1, uniformly stirring, placing in a vacuum drier, and exhausting by using a negative pressure vacuum pump until no bubbles exist;
pouring PDMS glue on the micro-column array silicon template 6, placing the micro-column array silicon template in an oven when the whole template is uniformly paved with the PDMS glue, and curing for 1h at 75 ℃ to obtain a PDMS three-dimensional micro-column array substrate 3; the protruding micro-pool on the micro-column array silicon template 6 is circular, the diameter is 5 μm, the height is about 500nm, and the distance between the micro-pool and the center of the micro-pool is 10 μm;
and step three, pouring PDMS glue on the microporous array perforated membrane silicon template 7, homogenizing at 1400rpm for 60s, and curing at 75 ℃ for 1h to obtain the PDMS microporous array perforated membrane 4. The silicon template 7 comprises 7 × 13 square units, each square unit consists of 10 × 10 square micro-columns, the side length of each micro-column is 100 μm, the height of each micro-column is about 150 μm, the distance between the centers of every two adjacent micro-columns is 200 μm, and the distance between the centers of every two adjacent square units is 3.0 mm;
and step four, peeling the three-dimensional micro-column array PDMS substrate and the microporous array perforated film from the template respectively, and aligning and flatly paving the microporous array perforated film and the three-dimensional micro-column array PDMS substrate along the edge. Obtaining the micro-pore array PDMS chip 2 with the three-dimensional micro-column array as the substrate.
The prepared chip is shown in fig. 5-6, wherein fig. 5A is a 10-fold bright field micrograph of the PDMS micro-column array, and B is a 40-fold bright field magnified image of the labeled area of fig. a. A scale: 50 μm. Fig. 5 demonstrates that the fabricated microcolumns are uniformly distributed in an array, and fig. 6 illustrates that a micro-pore array PDMS chip based on a three-dimensional microcolumn array is obtained, and a micro-pore array perforated membrane provides sufficient micro-chamber space for cell culture.
Example 2
The chip in the embodiment is applied to cell culture on a three-dimensional micro-column array substrate, and human breast tumor cells (MCF-7) are cultured on a three-dimensional micro-column array PDMS substrate (M-pilar) and a two-dimensional micro-column-free PDMS substrate (P-PDMS). The method specifically comprises the following steps:
the method comprises the following steps: respectively preparing a piece of three-dimensional micro-column array substrate PDMS and a PDMS chip without micro-columns, wherein the preparation process of the three-dimensional micro-column array substrate PDMS is as in embodiment 1, and the chip is subjected to hydrophilic treatment for 1min by plasma;
step two: mixing the above two cells at 5 × 104cells/mL, 200 mu L/well are respectively inoculated on a three-dimensional micro-column array substrate PDMS chip and a PDMS chip without micro-columns;
step three: 37 ℃ and 5% CO2Culturing for 24h in an incubator; staining cells with Calcein (Calcein-AM) for 30min, discarding staining agent, and washing with PBS for 3 times;
step four: and (5) taking pictures by microscope bright field and fluorescence, and recording the cell morphology.
The results of this example are shown in FIG. 7, which shows that the spread area of MCF-7 cells on the three-dimensional micro-column array substrate is smaller and the cell morphology tends to be circular compared to the PDMS substrate without the micro-column array.
Example 3
The chip in this embodiment is applied to three-dimensional co-culture of cells, and the structure of the chip is shown in fig. 1-4, the chip comprises a three-layer structure, which is a biological analysis substrate 1 from top to bottom in sequence, a micro-pore array chip 2 of the three-dimensional micro-column substrate comprises a middle-layer micro-pore array perforated membrane 3 and a lower-layer three-dimensional micro-column array substrate 4, and the micro-pore array chip is completed by flatly paving the micro-pore perforated membrane on the micro-column array substrate.
The biological analysis chip 2 is a micropore array PDMS chip with a three-dimensional microcolumn substrate.
In this embodiment, the micro-pore array PDMS chip of the three-dimensional micro-pillar substrate is fabricated by using the MEMS technology and the soft lithography method, and the fabrication steps and the physical diagram are shown in fig. 4 to 6, and the specific steps are as in example 1:
the chip in the embodiment is used for three-dimensional co-culture of PMA differentiated U937 macrophage and MCF-7 cells, and comprises the following steps:
the method comprises the following steps: carrying out hydrophilic treatment on a micro-pore array PDMS chip plasma of the three-dimensional micro-column substrate for 2 min;
step two: calcein-labeled U937 macrophages (1 × 10)4cells/mL) and MCF-7 tumor cells (1X 10)5cells/mL) as 1: 1 volume ratio in the chip;
step three: 37 ℃ and 5% CO2Culturing for 16h in an incubator;
step four: and (5) taking pictures by scanning a microscope bright field and a fluorescence large image, and recording the distribution of the cells in the holes.
The results of this example are shown in FIGS. 8-10, which indicate that the chip realizes three-dimensional co-culture of cells, and U937 macrophages and MCF-7 tumor cells in the wells are Gaussian-distributed. The number of MCF-7 in the single U937 macrophage hole in the hole is different, so that different tumor microenvironments are provided, and the cells keep good activity after being cultured for 16h, and can be used for subsequent multi-index detection and analysis.
Example 4
The chip in this example was applied to multi-index biomolecular analysis under three-dimensional co-culture of cells, here for the detection of two secreted proteins. The chip structure is as described in example 1, and the specific steps of cell seeding are as described in example 3.
The bioassay substrate is an antibody-modified polylysine slide.
In this embodiment, the PDMS micro-pore array chip with the three-dimensional micro-column as the substrate is fabricated by using the MEMS technology and the soft lithography method, as shown in fig. 1 to 4, and the specific steps are as in example 1:
the chip in the embodiment is applied to analysis of various secreted proteins of U937 macrophage single cell and MCF-7 three-dimensional co-culture, and specifically comprises the following steps:
the method comprises the following steps: polylysine slide (bioassay substrate) coated with TNF-a, IL-6 antibody;
step two: carrying out hydrophilic treatment on the three-dimensional micro-column array substrate and the micro-pore array chip plasma without the micro-column array substrate for 2 min;
step three: calcein-labeled U937 macrophages (1 × 10)4cells/mL) and MCF-7 (1X 10)5cells/mL) as 1: 1 volume ratio on the chip;
step four: 37 ℃ and 5% CO2Culturing for 3h in an incubator;
step five: covering the biological analysis substrate obtained in the first step, fixing a clamp, and culturing for 12h in an incubator;
step six: scanning and taking a picture by a microscope bright field and a fluorescence large image;
step seven: the fixture was removed, the slide was subjected to subsequent ELISA detection, the scanner scanned the slide, and secreted protein signals were recorded. The results of this example are shown in FIGS. 11-12, which show that the three-dimensional substrate inhibits TNF-a secretion from U937 macrophages, a single cell, while IL-6 secretion is slightly increased. In addition, the MCF-7 tumor microenvironment regulates TNF-a and IL-6 secretion differently, i.e., the MCF-7 tumor microenvironment inhibits TNF-a secretion without significant changes in IL-6 secretion rates.
It will be apparent to those skilled in the art from this disclosure that many changes and modifications can be made, or equivalents modified, in the embodiments of the invention without departing from the scope of the invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention shall still fall within the protection scope of the technical solution of the present invention, unless the contents of the technical solution of the present invention are departed.
Claims (10)
1. The micro-pore array chip of the three-dimensional micro-column substrate is characterized by comprising a substrate (3) and a micro-pore array perforated membrane (4), wherein the substrate (3) is the micro-column array substrate.
2. The three-dimensional micro-column substrate micro-well array chip of claim 1, wherein the cross-section of the micro-column is circular, regular polygonal or rectangular, and the area of the cross-section of the micro-column is 70 x 10-6μm2-70μm2The height of the microcolumn is 100nm-1cm, and the distance between the boundaries of two adjacent microcolumnsIs 100nm-1 cm.
3. The three-dimensional microcolumn-based microwell array chip of claim 1, wherein the microwell array perforated film (4) is composed of 1 or more microwell arrays, each microwell array having a shape of a regular polygon, a rectangle or a circle, the microwells (8) having a shape of a circle, a regular polygon or a rectangle, and the cross-sectional area of the microwells is 0.5 μm2-2×105μm2The distance between the boundaries of two adjacent micropores is 100 mu m-1 mm; the thickness of the micropore array perforated membrane (4) is 1 mu m-1 mm.
4. The micro pore array chip of the three-dimensional micro column substrate according to claim 1, wherein the substrate (3) and the micro pore array perforated membrane (4) are made of one or more composite materials selected from PDMS, PMMA, PC, PS, POE, EVA, EPDM, or one or more composite materials selected from PDMS, PMMA, PC, PS, POE, EVA, EPDM after hydrophilic modification.
5. A bioassay chip for realizing three-dimensional co-culture of cells, wherein the chip comprises a bioassay substrate and the chip of any one of claims 1 to 4, wherein the bioassay substrate is connected to the microporous array perforated membrane.
6. The biological analysis chip capable of realizing three-dimensional co-culture of cells according to claim 5, wherein the material of the biological analysis substrate (1) is polylysine, epoxy resin, amino-aldehyde modified glass or epoxy resin glass slide.
7. The bioassay chip as set forth in claim 6, wherein said glass or epoxy resin slide contains coated antibodies, proteins or DNA or coated protein arrays, antibody arrays or DNA arrays.
8. The use of the three-dimensional microcolumn-based microwell array chip of claim 1, wherein the chip is used for three-dimensional culture or co-culture of one or more single cells; the chip is applied to three-dimensional co-culture of one or more kinds of multiple cells.
9. The application of the chip for multi-index biological analysis capable of realizing three-dimensional co-culture of cells as claimed in claim 1, wherein the application is single-cell and multi-cell three-dimensional co-culture analysis of various cells, single-cell multi-biomolecule analysis of various cells, and multi-biomolecule analysis of various cells.
10. The use of claim 9, wherein said multiplex biomolecular analysis comprises analysis of secreted proteins, cytokines, growth factors, cell surface proteins, cell surface receptors, polypeptides, nucleic acids, hormones, antigens or exosomes, and said multiplex analysis comprises analysis of 1 to 100 indices.
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| CN202010774074.6A CN114058502A (en) | 2020-08-04 | 2020-08-04 | Biological analysis chip capable of realizing three-dimensional co-culture and multi-index detection of cells |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100129908A1 (en) * | 2008-11-21 | 2010-05-27 | Ye Fang | Spaced projection substrates and devices for cell culture |
| CN103122311A (en) * | 2013-01-15 | 2013-05-29 | 西北工业大学 | Flexible three-dimensional single-cell targeted cultivating chip and controllable preparation method thereof |
| CN109810895A (en) * | 2019-02-27 | 2019-05-28 | 西北工业大学 | Open three dimensional cell cultivation chip and its technology of preparing based on contour microtrabeculae |
| CN110343655A (en) * | 2018-04-02 | 2019-10-18 | 中国科学院大连化学物理研究所 | A kind of chip of integrating cell capture, three-dimensional substrates culture and bioanalysis |
-
2020
- 2020-08-04 CN CN202010774074.6A patent/CN114058502A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100129908A1 (en) * | 2008-11-21 | 2010-05-27 | Ye Fang | Spaced projection substrates and devices for cell culture |
| CN103122311A (en) * | 2013-01-15 | 2013-05-29 | 西北工业大学 | Flexible three-dimensional single-cell targeted cultivating chip and controllable preparation method thereof |
| CN110343655A (en) * | 2018-04-02 | 2019-10-18 | 中国科学院大连化学物理研究所 | A kind of chip of integrating cell capture, three-dimensional substrates culture and bioanalysis |
| CN109810895A (en) * | 2019-02-27 | 2019-05-28 | 西北工业大学 | Open three dimensional cell cultivation chip and its technology of preparing based on contour microtrabeculae |
Non-Patent Citations (3)
| Title |
|---|
| MENEKSE ERMIS: "A high throughput approach for analysis of cell nuclear deformability at single cell level", SCIENTIFIC REPORTS, vol. 6, pages 2 * |
| 刘军山等: "用于细胞三维培养的集成微柱阵列的微流控芯片设计与验证", 《分析化学》 * |
| 刘军山等: "用于细胞三维培养的集成微柱阵列的微流控芯片设计与验证", 《分析化学》, no. 08, 31 December 2017 (2017-12-31), pages 16 - 22 * |
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