CN111269873A - 3D islet cell vascularization microsphere construction method based on micro-pit chip - Google Patents
3D islet cell vascularization microsphere construction method based on micro-pit chip Download PDFInfo
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Abstract
The invention provides a 3D islet cell vascularization microsphere construction method based on a micro-pit chip, which utilizes a soft lithography method to prepare a micro-pit array template with a pyramid shape, and then Polydimethylsiloxane (PDMS) is used to prepare a micro-pit array chip with an inverse pyramid structure, wherein islet cells (β -TC6) and islet endothelial cells (MS-1) are inoculated on the micro-pit chip, the micro-pits have inclined side walls to force the cells to gather at the bottom of the micro-pits, and further gather into a three-dimensional islet cell mass.
Description
Technical Field
The invention belongs to the fields of microfluidic technology and cell biology, and particularly relates to a 3D islet cell vascularization microsphere construction method based on a micro-pit chip.
Background
The micro-fluidic chip laboratory is a technical platform which integrates basic operation units of sample preparation, reaction, separation, detection, cell culture, separation, division and the like in the fields of chemistry, biology and the like on a chip with the square centimeter (even smaller), and a network is formed by micro-channels so that controllable fluid penetrates through the whole system to replace various functions of the conventional chemical and biological laboratories. The basic characteristic and the greatest advantage of the microfluidic chip are that various unit technologies are flexibly combined and integrated on a scale on an integrally controllable micro platform. High throughput is a form of scale integration, except that the operating units or groups of units being integrated are the same.
With the development of cell biology and tissue engineering, three-dimensional cell culture technology is gradually replacing the traditional two-dimensional cell culture technology. At present, various types of cells have strong self-assembly capacity, such as pluripotent stem cells, tumor cells, tissue cells and the like. The three-dimensional cell spheres are three-dimensional aggregates formed by self-assembly of various cells, are closer to the structural morphology of in vivo tissue cells and are more beneficial to the research of functional mechanisms of the in vivo tissue cells. Thus, three-dimensional cell spheres can be used in numerous biological and biomedical fields of research, such as: developmental, pathological, pharmacological, cancer treatment, etc.
The existing three-dimensional cell aggregation culture methods mainly comprise a suspension drop method, suspension culture, a liquid drop method and the like, and the controllability and the repeatability of cell balls prepared by the methods are poor. In contrast, the chip with the concave structure can realize high throughput and higher controllability and repeatability when used for cell aggregation culture.
At present, most chips with a concave structure have vertical side walls, and cells settle at the bottom of the concave structure under the action of gravity to gather. However, such a recessed structure is disadvantageous to some extent in promoting cell aggregation. Because the cell mass is directly contacted with the bottom of the concave structure, the cell mass can not be fully contacted with nutrient substances and oxygen, most dead cells can be gathered on the side wall and the bottom, and the activity and the function of the cell balls are further influenced.
The invention utilizes a micro-pit chip with an inclined side wall to perform islet cell aggregation culture. The inclined side walls exert a certain physical mechanical force on the cells, which helps to promote the formation of cell balls under the combined action of gravity. Meanwhile, the three-dimensional structure reduces the contact area of the cell ball and the bottom, and maintains the better activity and function of the cell ball.
Disclosure of Invention
The invention aims to provide a 3D islet cell vascularization microsphere construction method based on a micro-pit chip, which aims to solve the problems that the physiological environment of cells in vivo cannot be simulated in the conventional 2D cell culture and the like.
In order to solve the problems, the invention provides a method for constructing 3D islet cell vascularization microspheres based on a micro-pit chip.
The preparation method of the micro-pit chip comprises the following specific steps:
(1) spin-coating SU-83035 photoresist with the thickness of 300-900 microns on the surface of the silicon wafer, and baking for 3-6 hours at the temperature of 95 ℃;
(2) fixing a square array mask with the side length of 200-400 microns on the surface of a substrate containing photoresist, and placing the square array mask on a platform which can adjust the inclination angle and can rotate freely;
(3) during ultraviolet exposure, the inclination angle (10-80 degrees) of the platform can be adjusted, and after each exposure, the platform can rotate at a rotation angle of 45 degrees or 90 degrees and then carry out the next exposure;
(4) baking for 10-30 minutes at 95 ℃, naturally cooling, and dissolving the unexposed SU-8 photoresist by using ethyl lactate to form a pyramid-shaped convex array template;
(5) hardening the film for 2 hours at 180 ℃, and finally preparing the micro-pit chip by back molding the PDMS.
The preparation method of the three-dimensional islet cell vascularization microsphere comprises the specific steps of inoculating β -TC6 islet cells and MS-1 islet endothelial cells into a PDMS chip with a micro-pit structure with an inclined side wall according to the volume ratio of 0.1-5: 1, gathering the cells at the bottom of a concave structure under the combined action of mechanical force and gravity generated by the inclined side wall, and self-assembling to form the three-dimensional islet cell vascularization microsphere, wherein the cell inoculation density is 105～106And (2) adding a culture medium into the micro-pit chip for culture for 12-24 hours per mL. The culture medium is a DMEM high-sugar culture medium which contains 15% of fetal calf serum, 1% of glutamine and 1% of double antibody in percentage by mass.
The micro-pit chip is composed of a single-layer open PDMS chip, wherein the number of the micro-pits is 50-300, and the size of the PDMS chip is 1-3 cm in length, 1-3 cm in width and 1-2 mm in height.
The micro-pit of the micro-pit chip is obtained by adjusting the inclination angle of the exposure platform to form a platform capable of freely rotating and performing ultraviolet exposure by using soft lithography.
The bottom surface of each micro pit of the micro pit chip is square, the side length is 100-300 mu m, the top of each micro pit is a square opening, the side length is 300-900 mu m, the height of each micro pit is 300-900 mu m, and the unit interval of each micro pit is 50-200 mu m.
The size of the three-dimensional islet cell vascularization microsphere formed by the micro-pit chip is 100-600 mu m.
An application of a 3D islet cell vascularization microsphere construction method based on a micro-pit chip in any one of the following items:
1) inducing and differentiating stem cells;
2) screening drugs;
3) establishing a physiological and pathological model;
4) culturing tumor cells;
5) culturing tissue cells;
6) high-throughput cellular microspheres;
7) in situ cell culture, administration, proliferation, induced differentiation and functional expression.
When the micro-pit chip is exposed by using soft lithography, the inclination angle of the exposure platform can be adjusted to form a platform capable of freely rotating, and a concave micro-pit with an inclined side wall is formed after exposure.
The method can be used with single or multiple cells, for example: stem cells, tumor cells, tissue cells, and the like. The three-dimensional cell microsphere formed by the method has better uniformity and high flux, and can be applied to various biological field researches after the three-dimensional cell microsphere is formed, such as: stem cell induced differentiation, drug screening, establishment of physiological and pathological models and the like.
The micro-pit chip can be used for inoculating different types of cells, and the cell inoculation density is 105～106And each/mL, after the micro-pit chip is added with a culture medium and cultured for 12-24 hours, cells can be gathered at the bottom of the micro-pit to form cell microspheres. The cell microspheres formed by the chip can be continuously cultured on the chip or taken out and then cultured separately.
Each micro-pit of the micro-pit chip can only form one cell microsphere, and the size of the micro-pit can meet the long-term growth and proliferation of cells.
The invention has the advantages that:
1. the operation is simple, convenient and quick;
2. the dosage of cells and reagents is small, and the experiment cost is low;
3. high integration;
4. the application range is wide;
drawings
In order to more clearly illustrate the technical scheme of the invention, the drawings used in the technical scheme description are simply introduced.
FIG. 1 is a schematic view of a micro-pit chip structure.
FIG. 2 is a schematic diagram of three-dimensional cell aggregation culture.
FIG. 3 islet cell vascularization microspheres formed in the micro-pit chip (7 days).
FIG. 4 proliferation of vascularized microspheres of islet cells in a micro-well chip (7 days).
FIG. 5 quantitative detection of insulin secretion from the vascularized microspheres of islet cells in the micro-pit chip (days 1, 4, 7).
Detailed Description
The present invention is further described with reference to the following specific examples, but the scope of the present invention is not limited by the examples, and if one skilled in the art makes some insubstantial modifications and adaptations to the present invention based on the above disclosure, the present invention still falls within the scope of the present invention.
A3D islet cell vascularization microsphere construction method based on a micro-pit chip is characterized in that a 3D islet vascularization microsphere is formed by each micro-pit, the microsphere generation is flexible and controllable, the flux is high, the separation efficiency of the single cell microsphere is high, the micro-pit structure is more beneficial to full exchange of nutrient substances, and the activity and the function of the islet vascularization microsphere are maintained.
Example 1
Preparation of micro-pit chip for 3D islet cell vascularization microsphere formation
Spin-coating SU-83035 photoresist with the thickness of 500 μm on the surface of a circular polished silicon wafer with the diameter of 50mm, and baking for 3 hours at the temperature of 95 ℃; fixing a square array mask with the side length of 300 microns on the surface of a substrate containing photoresist, and placing the square array mask on a platform which can adjust the inclination angle and can rotate freely; during ultraviolet exposure, adjusting the inclination angle of the platform to be 45 degrees, and simultaneously carrying out next exposure after the platform rotates at a rotation angle of 45 degrees after each exposure; baking for 30 minutes at 95 ℃, naturally cooling, and dissolving the unexposed SU-8 photoresist by using ethyl lactate to form a pyramid-shaped convex array template; hardening the film for 2 hours at 180 ℃, and finally preparing the micro-pit chip by back molding the PDMS.
Example 2:
preparation of 3D islet cell vascularization microspheres based on micro-pit chip
The periphery of the PDMS micro-pit chip prepared in the above example 1 was sealed with a 1cm high quartz ring through the top and bottom to form a micro-pit chamber with an open space, and as shown in FIG. 3, islet cells (β -TC6) and islet endothelial cells (MS-1) single cell suspension were seeded into the micro-pit chip at a volume ratio of 1:3, 1:1, 3:1, and the final seeding density was 2 × 105And each well, after the micro-pit chip is added with islet cell culture medium (high-glucose DMEM + 15% FBS + 1% Glutamine + 1% double antibody) and cultured for 24 hours, the cells can aggregate at the bottom of the micro-pit to form cell microspheres as shown in FIG. 2. Culturing for 24h, gently sucking away cell waste liquid and non-agglomerated scattered cells by using a pipette, supplementing a fresh culture medium, and continuously culturing the islet cells and the islet endothelial cell composite microspheres on the chip for 7 days; culturing the cells for 1, 4 and 7 days, respectively collecting culture media, and detecting the insulin secretion level by using an insulin secretion kit; and (4) culturing the cells for 7 days, adding a dead and live staining reagent DAPI-PI, and identifying the cell survival rate.
As shown in figure 1, the prepared micro-pit chip has a fixed size, the bottom side of the micro-pit is 200 microns, the top side is 500 microns, the size is uniform, the first day of cell culture is as shown in figures 2 and 3, islet cell nuclei and islet endothelial cells aggregate into spheres, the growth state is good, the light transmittance of the cell spheres is good, the cell survival rate is high, most cells survive and only a small amount of cells die after the seventh day of cell culture as shown in figure 4, the cell growth and proliferation condition is good, the higher the proportion of the islet endothelial cells is, the higher the cell survival rate is, and the insulin secretion detection of the cells cultured for 1, 4 and 7 days by using an insulin detection kit is as shown in figure 5, and the microspheres in the group of β -TC6 MS-1 cells (3:1) can secrete insulin most.
Claims (10)
1. A3D islet cell vascularization microsphere construction method based on a micro-pit chip is characterized in that: the method mainly comprises the following steps:
(1) preparing a micro-pit chip;
(2) preparing three-dimensional islet cell vascularization microspheres.
2. The method for constructing the 3D islet cell vascularization microspheres based on the micro-pit chip according to claim 1, is characterized in that: the micro-pit chip is composed of a single-layer open PDMS chip, and the number of the micro-pits is 50-300, wherein the micro-pit chip is provided with sunken micro-pits;
the PDMS chip is 1-3 cm long, 1-3 cm wide and 1-2 mm high.
3. The method for constructing the 3D islet cell vascularization microspheres based on the micro-pit chip according to claim 2, is characterized in that: the micro-pit of the micro-pit chip is obtained by adjusting the inclination angle of the exposure platform to form a platform capable of freely rotating and performing ultraviolet exposure by using soft lithography.
4. The method for constructing the 3D islet cell vascularization microspheres based on the micro-pit chip according to claim 2, is characterized in that: the bottom surface of each micro pit of the micro pit chip is square, the side length is 100-300 mu m, the top of each micro pit is a square opening, the side length is 300-900 mu m, the height of each micro pit is 300-900 mu m, and the unit interval of each micro pit is 50-200 mu m.
5. The method for constructing the 3D islet cell vascularization microspheres based on the micro-pit chip according to claim 1, is characterized in that: the preparation method of the micro-pit chip in the step (1) comprises the following steps:
(1) spin-coating SU-83035 photoresist with the thickness of 300-900 microns on the surface of the silicon wafer, and baking for 3-6 hours at the temperature of 95 ℃;
(2) fixing a square array mask with the side length of 200-400 microns on the surface of a substrate containing photoresist, and placing the square array mask on a platform which can adjust the inclination angle and can rotate freely;
(3) during ultraviolet exposure, the inclination angle of the platform can be adjusted to 10-80 degrees, and after each exposure, the platform can rotate at a rotation angle of 45 degrees or 90 degrees and then carry out the next exposure;
(4) baking for 10-30 minutes at 95 ℃, naturally cooling, and dissolving the unexposed SU-8 photoresist by using ethyl lactate to form a pyramid-shaped convex array template;
(5) hardening the film for 2 hours at 180 ℃, and finally preparing the micro-pit chip by back molding the PDMS.
6. The method for constructing the 3D islet cell vascularization microspheres based on the micro-pit chip according to claim 1, wherein the three-dimensional islet cell vascularization microspheres of the step (2) are prepared by inoculating β -TC6 islet cells and MS-1 islet endothelial cells into a PDMS chip of a micro-pit structure with an inclined side wall according to a volume ratio of 0.1-5: 1, and under the combined action of mechanical force and gravity generated by the inclined side wall, the cells are gathered at the bottom of a concave structure and self-assembled to form the three-dimensional islet cell vascularization microspheres.
7. The method for constructing the 3D islet cell vascularization microspheres based on the micro-pit chip according to claim 6, is characterized in that: the size of the three-dimensional islet cell vascularization microsphere formed by the micro-pit chip is 100-600 mu m.
8. The method for constructing the 3D islet cell vascularization microspheres based on the micro-pit chip according to claim 6, is characterized in that: in the preparation process of the three-dimensional islet cell vascularization microsphere in the step (2), the cell inoculation density is 105～106And (2) adding a culture medium into the micro-pit chip for culture for 12-24 hours per mL.
9. The method for constructing the 3D islet cell vascularization microspheres based on the micro-pit chip according to claim 8, is characterized in that: the culture medium in the step (2) is a DMEM high-sugar culture medium, wherein the DMEM high-sugar culture medium contains 15% of fetal calf serum, 1% of glutamine and 1% of double antibodies in percentage by mass.
10. The use of the micro-pit chip based 3D islet cell vascularization microsphere construction method according to claims 1-9 in any one of the following:
1) inducing and differentiating stem cells;
2) screening drugs;
3) establishing a physiological and pathological model;
4) culturing tumor cells;
5) culturing tissue cells;
6) high-throughput cellular microspheres;
7) in situ cell culture, administration, proliferation, induced differentiation and functional expression.
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