CN118272231A - Pig endometrium micro-fluidic chip and preparation method and application thereof - Google Patents
Pig endometrium micro-fluidic chip and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of organ chips and discloses a pig endometrium microfluidic chip and a preparation method and application thereof, wherein the chip comprises a chip main body, the chip main body is of a concave structure formed by protruding at two sides of a concave part in the middle, an upper side plate is embedded in the concave part, a bottom gel pool is arranged in the concave part, a top liquid storage pool penetrating through the upper side plate is arranged on the upper side plate, the top liquid storage pool and the bottom gel pool are mutually communicated, a gel bracket is filled in the bottom gel pool, and a bionic uterine gland structure is arranged on the surface of the gel bracket; the bulge is provided with a plurality of groups of pouring liquid storage pool groups, each group of pouring liquid storage pool group consists of two pouring liquid storage pools which are respectively arranged on the bulge, and the two pouring liquid storage pools are symmetrically arranged relative to the concave part and are communicated through a pipe cavity, and the pipe cavity penetrates through the gel bracket. The pig endometrium micro-fluidic chip can truly simulate functions of cell composition and the like of pig endometrium in vitro, and realizes microenvironment simulation of uterine cavity through co-culture.
Description
Technical Field
The invention relates to the technical field of organ chips, in particular to a pig endometrium micro-fluidic chip and a preparation method and application thereof.
Background
With the rapid development of the economy in China, the population is numerous, and the livestock industry is also rapidly developing. Pigs are important agricultural farmed animals, and their reproductive capacity and farrowing rate directly affect the development and economic benefits of animal husbandry. Due to the increasing pork demand, it is necessary to increase the productivity of the pig industry, starting from the productivity of sows. Endometrium is a critical tissue controlling the reproductive process of pigs and plays an important role in embryo implantation, placenta formation and fetal development. Studying the structure, function and metabolic regulation of pig endometrium can help us to better understand the physiological process of pig reproduction and the mechanism of reproductive disorder. Through further research on the relationship between the change of the endometrium of the pig and the health and reproductive performance, a powerful scientific basis can be provided for improving the reproductive rate of the sow and improving the productivity.
Currently, in existing methods for studying pig endometrium, living animals (such as pigs or mice) are generally used as a study model, and physiological and pathological changes and mechanisms of pig endometrium are studied mainly by methods such as but not limited to clinical symptom observation, histological examination, molecular biological detection, multi-group chemical combined analysis and the like. However, the use of living animals, particularly sow-based tests, has ethical problems of high cost, long period, low repeatability and unavoidable avoidance; the research by taking mice as models faces the difference among different species, so that some results are difficult to verify in pigs; in addition, in vivo experiments also have difficulty in achieving real-time dynamic observation of endometrial tissue development and regulation processes.
The in vitro cell model has the advantages of simple operation, low cost and the like, and by utilizing the in vitro cell culture system, the generation and change process of the endometrium of the pig can be simulated by using the endometrial tissue or cells of the pig, so that the experimental condition can be better controlled, and the repeatability of the experiment can be improved. However, the cell type of the existing pig endometrium in-vitro cell model is single, and complex and various cell compositions and three-dimensional structures of the pig endometrium are difficult to reproduce, so that in-vitro simulation and in-vivo real physiological states are greatly different. Therefore, a new technical method is needed to realize the composition and structural functions of pig endometrial cells and overcome the limitations of the model.
The organ chip is to construct an organ physiological microenvironment containing various living cells, functional tissue interfaces, biological fluid, mechanical force stimulation and other complex factors on the microfluidic chip, simulate an in-vitro model of the main structure and functional characteristics of tissue and organs, and currently, the research on embryo development by utilizing the organ chip technology is mainly carried out aiming at human and mice, so that the research on the attached implantation of embryo development of pigs is blank. The embryo development and implantation process of human and mice is greatly different from that of pigs, and a pig uterine chip model for researching the embryo implantation process is not established yet.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a pig endometrium micro-fluidic chip, a preparation method and application thereof. The chip can be used for intuitively observing the attachment process of the porcine embryo trophoblast cells on the endometrium and can be used for researching the regulation and control of microorganism metabolites on the embryo attachment process.
In order to achieve the above purpose, the present invention provides a pig endometrium micro-fluidic chip, which comprises a chip main body, wherein the chip main body is a concave structure formed by protruding at two sides of a concave part in the middle, an upper side plate is embedded in the concave part of the chip main body, a bottom gel pool is arranged on the concave part of the chip main body, a top liquid storage pool penetrating through the upper side plate is arranged on the upper side plate, the top liquid storage pool and the bottom gel pool are mutually communicated, a gel bracket is filled in the bottom gel pool, and a bionic uterine gland structure is arranged on the surface of the gel bracket;
The convex part of the chip main body is provided with a plurality of groups of pouring liquid storage tank groups, each group of pouring liquid storage tank group consists of two pouring liquid storage tanks which are respectively arranged on the convex part of the chip main body, and the two pouring liquid storage tanks are symmetrically arranged relative to the concave part;
wherein, two perfusion liquid storage tanks which are symmetrically arranged relative to the concave part are communicated through a pipe cavity, and the pipe cavity penetrates through the gel bracket.
Preferably, the gel scaffold is formed by curing a hydrogel or a mixture containing hydrogels.
Further preferably, the hydrogel-containing mixture further comprises stromal cells and/or immune cells.
Preferably, the density of cells in the hydrogel-containing mixture is 2X 10 5～2×107 cells/mL.
Further, when the gel scaffold is formed by solidifying a mixture containing hydrogel and stromal cells, the preparation method of the gel scaffold comprises: mixing the matrix cells with the hydrogel to obtain a mixture containing the hydrogel and the matrix cells, and incubating for 20-30min to solidify the mixture containing the hydrogel and the matrix cells.
Preferably, the bionic uterine gland structure is a micro-concave array structure.
Further preferably, the biomimetic uterine gland structure occupies 40% and more of the surface area of the gel scaffold.
Preferably, each pouring liquid storage tank is of a cuboid structure, the height is 3-6mm, the length is 5-10mm, and the width is 2-5mm.
Preferably, the bottom gel tank is of a regular hexagonal prism structure, the side length is 4-8mm, and the height is 1-3mm.
Further preferably, the plurality of lumens are all arranged in parallel, and each lumen has a length of 10-20mm.
Further preferably, the pore diameter of the portion of each lumen located in the gel holder is 0.4-1mm, and the pore diameter of the portion located in the chip body is 0.8-1.5mm.
Further preferably, the height of the gel scaffold is 2-4mm.
Preferably, the top liquid storage tank is of a regular hexagonal prism structure, the cross sections of the top liquid storage tank and the bottom gel tank are the same in size, and the height is 2-4mm.
The second aspect of the invention provides a method for preparing the pig endometrium micro-fluidic chip, which comprises the following steps:
S1, designing a pig endometrium micro-fluidic chip module structure, preparing a mould by using a3D printing instrument and a photoetching method, pouring a mixture of polydimethylsiloxane prepolymer and a curing agent, solidifying, demolding and cutting to obtain a chip main body, an upper side plate, a tube cavity column and a cover plate with a convex array structure, wherein the chip main body is of a concave structure formed by protruding at two sides of a concave part in the middle part, a bottom gel pool is formed in the concave part of the chip main body, a top liquid storage pool penetrating through the upper side plate is formed in the upper side plate, a plurality of groups of pouring liquid storage pool groups are formed in the protruding part of the chip main body, each group of pouring liquid storage pool group consists of two pouring liquid storage pools respectively formed in the protruding part of the chip main body, and the two pouring liquid storage pools are symmetrically arranged relative to the concave part;
S2, perforating the side wall of each pouring liquid storage tank to enable the pouring liquid storage tanks to be communicated with a bottom gel tank, injecting hydrogel or a mixture containing the hydrogel into the bottom gel tank, forming a gel bracket with a bionic uterine gland structure on the surface in the bottom gel tank by utilizing a lumen column and a cover plate with a convex array structure, forming a plurality of lumens simultaneously, and communicating two pouring liquid storage tanks symmetrically arranged relative to a concave part by the lumens, wherein the lumens penetrate through the gel bracket, then assembling with an upper side plate, enabling the concave part of a chip main body to be embedded with the upper side plate, and enabling the top liquid storage tank and the bottom gel tank to be mutually communicated.
Further preferably, in step S1, the weight ratio of the polydimethylsiloxane prepolymer to the curing agent is (8-12): 1.
Further preferably, the preparation method of the hydrogel in step S2 includes: h 2O：NaHCO3 solution: HEPES solution: 10 XDMEM: hydrogel monomer = 8:1:1:10:80-180 volume ratio.
Further preferably, the hydrogel monomer is selected from one or more of bovine tail type i collagen, rat tail type i collagen and gelatin.
The third aspect of the invention provides an application of the pig endometrium micro-fluidic chip, wherein the application comprises culturing pig endometrium epithelial cells and pig endothelial cells, simulating the cell composition of pig uterus in vitro and simulating the three-dimensional structure of pig uterus in vitro.
The fourth aspect of the invention provides a use method of the pig endometrium micro-fluidic chip, which comprises the following steps:
(a) Injecting the pig endothelial cell suspension into each lumen of the chip, turning over four sides, standing for 20-40min respectively, enabling pig endothelial cells to closely adhere to and grow in a lumen channel of each lumen, then adding a complete culture medium into each perfusion liquid storage tank respectively, and standing for 20-30min;
Wherein the density of the porcine endothelial cell suspension is 1X 10 5～1×107/mL, and the dosage of single-lumen injection is 10-30 mu L;
(b) Injecting the pig endometrium epithelial cell suspension onto the gel bracket, standing and culturing for 20-40min, sucking the pig endometrium epithelial cell suspension, cleaning with PBS buffer solution, adding complete culture medium into the top liquid storage tank, and standing and culturing;
Wherein the density of the pig endometrium epithelial cell suspension is 1×10 4～1×106/mL, and the dosage of single injection is 100-200 μl.
The invention has the beneficial effects that:
(1) The pig endometrium micro-fluidic chip provided by the invention can truly simulate the cell composition of pig endometrium and simulate a three-dimensional uterine cavity structure in vitro, and realize the micro-environment simulation of the uterine cavity through co-culture.
(2) The pig endometrium micro-fluidic chip provided by the invention supports in-vitro co-culture of pig endometrium epithelial cells, endothelial cells and matrix cells, and can realize real-time observation and record of different cell growth states in uterus.
(3) The chip can be used for researching interaction between embryo trophoblast cells and pig endometrium, and screening nutrient substances or microorganism metabolites capable of promoting embryo attachment.
(4) The chip provided by the invention has the advantages of simple design, strong operability, good biocompatibility, low test cost, capability of greatly improving the test efficiency and meeting the requirement of large-scale experiments, and can truly simulate the pig uterus, and an in-vitro pig uterus simulation system which contains various cell types, has an epithelial-like villus structure, has a pourable vascular channel and has high physiological relevance is established in vitro, so that the problem that the pig reproductive system research lacks an in-vitro research model with high physiological relevance, high economic benefit and simple operation is solved, and an innovative research system based on organ level is provided for pig in-vitro embryo attachment, pig breeding and embryo culture and pig endometrium related research.
Drawings
FIG. 1 is a schematic diagram of a pig endometrium microfluidic chip;
FIG. 2 is a split view of a pig endometrium microfluidic chip;
FIG. 3 is a real object diagram of a pig endometrium micro-fluidic chip;
FIG. 4 is a flow chart of the fabrication of a pig endometrium microfluidic chip;
FIG. 5 is a diagram of pig endometrial epithelial cell culture within a pig endometrial microfluidic chip;
FIG. 6 is a diagram of a culture of porcine endometrial epithelial cells within a porcine endometrial microfluidic chip;
FIG. 7 is a diagram of pig endothelial cell culture in a pig endometrium microfluidic chip;
FIG. 8 is a graph of growth of stromal cells in a pig endometrium microfluidic chip;
FIG. 9 is a fluorescent staining chart of pig endometrial epithelial cell culture in a pig endometrial microfluidic chip;
FIG. 10 is a fluorescent staining chart of pig endothelial cell culture in a pig endometrium microfluidic chip;
FIG. 11 is a fluorescence plot of adhesion of porcine embryonic trophoblast cells on chip;
FIG. 12 is a fluorescence plot of adhesion of washed porcine embryonic trophoblast cells on chip;
FIG. 13 is a graph showing the results of the adhesion rate of porcine embryonic trophoblast cells on chip after culturing different cells.
Description of the reference numerals
1A chip main body; 2, an upper side plate; 3, a gel scaffold;
1.1 bottom gel cell; 1.2 a first lumen; 1.3 a second lumen; 1.4 a first priming reservoir;
1.5 a second priming reservoir; 1.6 third priming reservoir; 1.7 fourth priming reservoir;
2.1 a top reservoir; 3.1 bionic uterine gland structure.
Detailed Description
The present invention will be described in detail below with reference to the drawings and examples. It is to be understood that the embodiments described herein are for illustration and explanation of the invention, and are not intended to limit the invention.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
It should be noted that, if there is a directional indication (such as up, down, left, right, front, and rear …) in the embodiment of the present invention, the directional indication is merely used to explain the relative positional relationship, movement situation, etc. between the components in a specific posture (as shown in the drawings), and if the specific posture is changed, the directional indication is correspondingly changed.
In addition, if there is a description of "first", "second", etc. in the embodiments of the present invention, the description of "first", "second", etc. is for descriptive purposes only and is not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In addition, the technical solutions provided in the embodiments of the present invention may be combined with each other, but it is necessary to use those skilled in the art as a basis, and when the technical solutions are contradictory or cannot be implemented, it should be considered that the combination of the technical solutions does not exist and is not within the scope of protection claimed in the present invention.
Example 1
The pig endometrium micro-fluidic chip shown in fig. 1-3 comprises a chip main body 1, wherein the chip main body 1 is of a concave structure formed by protruding at two sides of a concave part in the middle, an upper side plate 2 is embedded in the concave part of the chip main body 1, a bottom gel tank 1.1 is arranged at the concave part of the chip main body 1, a top liquid storage tank 2.1 penetrating through the upper side plate is arranged at the upper side plate 2, the top liquid storage tank 2.1 and the bottom gel tank 1.1 are communicated with each other, a gel bracket 3 is filled in the bottom gel tank 1.1, and a bionic uterine gland structure 3.1 is arranged on the surface of the gel bracket 3;
The convex part of the chip main body 1 is provided with a plurality of groups of pouring liquid storage tank groups, each group of pouring liquid storage tank group consists of two pouring liquid storage tanks which are respectively arranged on the convex part of the chip main body 1, and the two pouring liquid storage tanks are symmetrically arranged relative to the concave part;
The two perfusion liquid reservoirs symmetrically arranged relative to the concave part are communicated through a pipe cavity, the pipe cavity penetrates through the gel bracket 3 (two pipe cavities (according with a physiological structure) are arranged in the embodiment, the two pipe cavities are respectively a first pipe cavity 1.2 and a second pipe cavity 1.3, the convex part of the chip main body 1 is provided with two groups of perfusion liquid reservoir groups, namely a first perfusion liquid reservoir group and a second perfusion liquid reservoir group, the first perfusion liquid reservoir group consists of a first perfusion liquid reservoir 1.4 and a third perfusion liquid reservoir 1.6 which are respectively arranged on the convex part of the chip main body 1, the first perfusion liquid reservoir 1.4 and the third perfusion liquid reservoir 1.6 are symmetrically arranged relative to the concave part, the second perfusion liquid reservoir group consists of a second perfusion liquid reservoir 1.5 and a fourth perfusion liquid reservoir 1.7 which are respectively arranged on the convex part of the chip main body 1, the second perfusion liquid reservoir 1.5 and the fourth perfusion liquid reservoir 1.7 are symmetrically arranged relative to the concave part, the first perfusion liquid reservoir 1.4 and the third perfusion liquid reservoir 1.6 which are symmetrically arranged relative to the concave part are respectively, and the first perfusion liquid reservoir 1.5 and the second perfusion liquid reservoir 1.6 are symmetrically arranged relative to the concave part, and the second perfusion liquid reservoir 1.1.7 and the second pipe cavity 1.3 is communicated through the pipe cavity 1.3 and the pipe cavity 1.6.
Further, the recessed portion of the chip main body 1 is embedded with the upper side plate 2 to form a cuboid structure, the length of the cuboid structure is 15-30mm, the width of the cuboid structure is 10-20mm, the height of the cuboid structure is 5-10mm, and further, in the embodiment, the length of the cuboid structure is 26mm, the width of the cuboid structure is 16mm, and the height of the cuboid structure is 8mm;
Wherein, the width of the upper side plate 2 is 8-15mm, the length is 12-24mm, the height is 2-5mm, in the embodiment, the width of the upper side plate 2 is 9mm, the length is 16mm, and the height is 4mm;
In the present invention, the gel-scaffold 3 is formed by curing a hydrogel or a mixture containing a hydrogel;
Wherein in the present embodiment, the gel scaffold 3 is formed by hydrogel curing;
In the technical scheme of the invention, the bionic uterine gland structure 3.1 is a micro-concave array structure, and the micro-concave array structure is formed by embedding a cover plate with a convex array structure into hydrogel or a mixture containing the hydrogel and then solidifying the cover plate; the micro-concave array structure is a structure with a regular cylinder concave array, the diameter of the cylinder is 50-200 mu m, the height of the cylinder is 100-300 mu m, the cylinders are arranged in an array manner, and the array period of the cylinders is 100-600 mu m; wherein in the present embodiment, the diameter of the cylinder is 200 μm, the height is 200 μm, and the array period of the cylinder is 200 μm;
In the invention, the bionic uterine gland structure 3.1 occupies 40% or more of the surface area of the gel support 3.
Further, in this embodiment, the biomimetic uterine gland structure 3.1 occupies 40% of the surface area of the gel-scaffold 3.
In the present invention, the gel scaffold 3 is a microenvironment for simulating endometrial extracellular matrix components and bearing cell growth, and the bionic uterine gland structure 3.1 on the surface is used for simulating the uterine gland structure of a pig.
The shape structures of the bottom gel tank 1.1, the first filling liquid storage tank 1.4, the second filling liquid storage tank 1.5, the third filling liquid storage tank 1.6 and the fourth filling liquid storage tank 1.7 in the invention can be any one of prismatic structures (the shape structures can be designed according to the functional requirements); the first perfusion liquid storage pool 1.4, the second perfusion liquid storage pool 1.5, the third perfusion liquid storage pool 1.6 and the fourth perfusion liquid storage pool 1.7 are all cuboid structures (used for containing culture medium, the culture mediums contained in the first perfusion liquid storage pool 1.4, the second perfusion liquid storage pool 1.5, the third perfusion liquid storage pool 1.6 and the fourth perfusion liquid storage pool 1.7 can be all the same), the heights are 3-8mm, the widths are 2-5mm, the lengths are 5-10mm, and further in the embodiment, the heights are 6mm, the lengths are 6mm, and the widths are 3mm; the bottom gel tank 1.1 is of a regular hexagonal prism structure, the side length is 4-8mm, the height is 1-3mm, and further in the embodiment, the side length is 4.4mm, and the height is 3mm;
The gel support 3 is tightly attached to the side wall of the bottom gel tank 1.1, and has a complete shape, the side length of the gel support 3 is 4-8mm, the height is 2-4mm, and further, in this embodiment, the side length of the gel support 3 is 4.4mm, and the height is 3mm, i.e., the side length of the gel support 3 is equal to the side length of the bottom gel tank 1.1;
The perfusion liquid storage tank groups of each group are composed of two perfusion liquid storage tanks which are respectively arranged on the convex parts of the chip main body, so that the purpose of being convenient for providing a circulating environment for cells inoculated in the lumen when the cells are inoculated in the lumen for culture in the follow-up process is achieved;
Further, the first lumen 1.2 and the second lumen 1.3 are arranged in parallel and used for simulating a blood vessel lumen, and the lengths of the first lumen 1.2 and the second lumen 1.3 are 10-20mm, and in the embodiment, the lengths of the first lumen 1.2 and the second lumen 1.3 are 15mm.
In the invention, the aperture of the part of the first lumen 1.2 and the second lumen 1.3 positioned in the gel bracket 3 is 0.4-1mm, and the aperture of the part of the first lumen 1.2 and the second lumen 1.3 positioned in the chip main body 1 is 0.8-1.5mm, so that a compact growth structure can be formed by planting cells in the lumen, and the size of a real blood vessel can be simulated;
In this embodiment, the apertures of the portions of the first lumen 1.2 and the second lumen 1.3 located in the gel support 3 are all 0.6mm, and the apertures of the portions of the first lumen 1.2 and the second lumen 1.3 located in the chip body 1 are all 1.0mm.
The purpose of the invention is to provide the upper side plate 2 and the top liquid storage tank 2.1 on the upper side plate 2, when the cells are cultured on the surface of the gel support 3 later, a certain amount of corresponding culture medium can be added and filled into the top liquid storage tank 2.1 to culture the cells, and meanwhile, the gel support 3 can be prevented from being directly contacted with the outside, and the gel support 3 is prevented from being polluted, dehydrated and dried.
Therefore, preferably, the top liquid storage tank 2.1 is in a regular hexagonal prism structure, the cross section of the top liquid storage tank 2.1 is the same as that of the bottom gel tank 1.1, and the height of the top liquid storage tank 2.1 is 2-4mm; the height of the top reservoir 2.1 in this embodiment is 4mm.
The physical diagram of the pig endometrium micro-fluidic chip is shown in fig. 3, wherein the chip main body 1 and the upper side plate 2 are made of Polydimethylsiloxane (PDMS), and PDMS is a material widely applied to microfluidics and organ chip preparation, and has good transparency, elasticity and biocompatibility; the processing is easy, and the flexibility and the plasticity are very high; the manufacturing cost is low; the properties of the PDMS film, such as hardness, adhesiveness and the like, can be controlled by adjusting the formula and the hardening condition of the PDMS film so as to adapt to the requirements of different applications; in addition, the hydrophilicity or hydrophobicity of the PDMS surface can be adjusted by surface treatment or coating, so that the control of the processes of liquid flow, cell attachment and the like can be realized. The hydrogel monomer is used as a material of the three-dimensional gel, has good biocompatibility, does not cause obvious immune reaction or rejection reaction, and is suitable for the fields of cell culture, tissue engineering, regenerative medicine and the like; can provide support for cell adhesion, promote the growth, migration and differentiation of cells in a three-dimensional structure, and is beneficial to tissue engineering and cell proliferation; and is degradable, without residue or causing chronic inflammatory reaction.
The preparation method of the pig endometrium micro-fluidic chip shown in fig. 4 comprises the following steps:
(1) The method comprises the steps of designing a pig endometrium micro-fluidic chip module structure (comprising designing a chip main body 1 and an upper side plate 2) by using Solidworks software, designing a structure of a tube cavity column, manufacturing a die of the chip main body 1, the upper side plate 2 and the tube cavity column by using a 3D printing instrument, and mixing a polydimethylsiloxane prepolymer and a curing agent according to a weight ratio of 8-12:1 (in this example, the ratio by weight of polydimethylsiloxane prepolymer to curing agent is 10:1, and the curing agent is purchased from Momentive, trade mark 9482) to obtain PDMS prepolymer; pouring the two parts on a die of a chip main body 1, an upper side plate 2 and a lumen column respectively, blowing off all bubbles on the surface by using an ear washing ball after vacuum treatment, performing heat treatment for 5h at 60-65 ℃ (the temperature of the heat treatment in the embodiment is 65 ℃) to solidify PDMS prepolymer, cutting the chip main body 1, the upper side plate 2 and the lumen column along the edge of the die by using a surgical knife after complete solidification, performing demolding treatment to finally obtain the chip main body 1, the upper side plate 2 and two lumen columns (the length of the lumen column is greater than the length of each lumen, the diameter of the lumen column is matched with the diameters of a first lumen 1.2 and a second lumen 1.3, the length is 18mm, the diameter is 0.6 mm), the chip main body 1 is a concave structure formed by the bulges at the two sides of a middle part depression, a bottom gel pool 1.1 is arranged at the depression part of the chip main body 1, a first perfusion pool group and a second perfusion pool group are arranged at the convex part of the chip main body 1, the first perfusion liquid storage pool group consists of a first perfusion liquid storage pool 1.4 and a third perfusion liquid storage pool 1.6 which are respectively arranged on the convex part of the chip main body 1, the first perfusion liquid storage pool 1.4 and the third perfusion liquid storage pool 1.6 are symmetrically arranged relative to the concave part, the second perfusion liquid storage pool group consists of a second perfusion liquid storage pool 1.5 and a fourth perfusion liquid storage pool 1.7 which are respectively arranged on the convex part of the chip main body 1, the second perfusion liquid storage pool 1.5 and the fourth perfusion liquid storage pool 1.7 are symmetrically arranged relative to the concave part, the top liquid storage pool 2.1 penetrating through the upper side plate 2 is arranged on the upper side plate 2, and the side walls of the first perfusion liquid storage pool 1.4, the second perfusion liquid storage pool 1.5, the third perfusion liquid storage pool 1.6 and the fourth perfusion liquid storage pool 1.7 are horizontally perforated by a puncher with an inner diameter of 1.0mm (for conveniently pumping a pipe column when a pipe cavity is formed later, the structure of the gel support 3 is not damaged, and the pore size of the hole is slightly larger than the size of the tube cavity column so as to be communicated with the bottom gel tank 1.1;
(2) Designing a mold with a convex array structure, manufacturing a convex substrate with a convex array by soft lithography, and processing the mold with the convex array structure: washing the silicon wafer with ethanol, washing with deionized water to remove pollutants on the surface of the silicon wafer, and finally dehydrating and drying; 1mL of SU8-2075 photoresist is dripped in the center of a silicon wafer, a spin coater is used for spin coating (the thickness of the photoresist spin coating is 200 mu m), the rotating speed is 1200rpm/min, and the time is 1min; performing pre-baking operation after gluing, placing on a heating plate at 65 ℃ for heating for 5min, and transferring to a heating plate at 98 ℃ for heating for 20min (the pre-baking function is to volatilize organic solvent in the photoresist, so that the photoresist on the surface of the silicon wafer is solidified, and the adhesiveness between the photoresist and the silicon wafer is increased); repeating the steps after the first photoresist layer is dried to carry out secondary photoresist coating, and carrying out secondary pre-drying according to the steps; covering a mask on the silicon wafer after the secondary pre-baking is finished, performing exposure treatment, manufacturing a convex array photoresist structure by using a photoetching machine and the mask, performing exposure twice, wherein the time of the two exposures is 5s, immediately performing post-baking after the exposure is finished, placing the silicon wafer on a hot plate at 65 ℃ for heating for 5min, transferring the silicon wafer on the hot plate at 95 ℃ for heating for 15min, performing development after cooling, removing the photoresist which is not solidified on the convex array structure on the silicon wafer by using a developing solution (propylene glycol monomethyl ether acetate), taking out the silicon wafer after the pattern is clear, flushing the silicon wafer by using isopropanol, and airing; finally, inverting the silicon wafer to carry out silanization overnight, wherein the reagent used in the silanization is trimethylchlorosilane, and preparing a die with a convex array structure;
(3) Pouring PDMS prepolymer on the mould with the convex array structure, solidifying, demoulding, cutting into 2mm PDMS cube blocks with the convex array structure, and bonding with a flat Polydimethylsiloxane (PDMS) sheet by oxygen plasma treatment: the method comprises the steps of respectively placing a PDMS cube block with a convex array structure and a flat Polydimethylsiloxane (PDMS) block which are prepared after solidification in a plasma instrument, rapidly attaching the PDMS cube block with the convex array structure to the center of the flat Polydimethylsiloxane (PDMS) sheet within 20s and 1 min after ignition, heating in a 60 ℃ oven for 10 min to enable bonding to be more complete, then punching two ends of the flat Polydimethylsiloxane (PDMS) sheet by using a 1mm puncher, respectively opening a gel sample inlet and a gel sample outlet, and finally obtaining a Polydimethylsiloxane (PDMS) cover plate with the convex array structure (by using the convex array structure on the cover plate, a corresponding micro-concave array structure can be formed on the surface of a gel bracket 3);
The Polydimethylsiloxane (PDMS) cover plate with the convex array structure is a convex structure formed by recessing two sides of a middle protrusion, and is prepared by bonding a PDMS square block with the convex array structure and a flat PDMS block (the flat PDMS block is of a cuboid structure, has a width of 8-15mm, a length of 12-24mm, and a height of 2-5mm, and further in the embodiment, the flat PDMS block has a width of 9mm, a length of 15mm, and a height of 3 mm), wherein the convex array structure is arranged on the surface of the convex portion, and a gel sample inlet and a gel sample outlet are respectively formed at two ends of the Polydimethylsiloxane (PDMS) cover plate with the convex array structure (if only one gel sample inlet can form a closed space, substances such as hydrogel cannot be injected into the gel sample inlet), and the recessed portion of the chip main body 1 can be assembled with the Polydimethylsiloxane (PDMS) cover plate with the convex array structure in a jogging mode;
(4) Chip assembly sterilization, modification and assembly: sterilizing and drying all the chip components to restore hydrophobicity; performing surface modification treatment on the bottom gel pool 1.1, immersing the bottom gel pool for 10min by using Polyethyleneimine (PEI), immersing the bottom gel pool for 30min by using Glutaraldehyde (GA) after removal, and then cleaning the bottom gel pool by using sterile water; then, a lumen column penetrates through a hole punched on the side wall of the pouring liquid storage tank to connect the pouring liquid storage tank on two sides with the bottom gel tank 1.1 (one lumen column penetrates through the hole on the side wall of the first pouring liquid storage tank 1.4, the hole on the side wall of the bottom gel tank 1.1 and the hole on the side wall of the third pouring liquid storage tank 1.6 in sequence, the other lumen column penetrates through the hole on the side wall of the second pouring liquid storage tank 1.5, the hole on the side wall of the bottom gel tank 1.1 and the hole on the side wall of the fourth pouring liquid storage tank 1.7 in sequence, and the two lumen columns are parallel), and a pair of tweezers are used for jogging and assembling the PDMS cover plate with the convex array structure and the concave part of the chip main body 1, so that the PDMS cover plate and the concave array structure on the PDMS cover plate are tightly attached (the convex array structure on the PDMS cover plate faces the bottom gel tank 1.1) to ensure that no leakage between components to obtain a first assembly for standby;
(5) Preparation of gel scaffold 3: selecting bovine tail type I collagen as a hydrogel monomer, and dissolving H 2O、NaHCO3 in a solution: HEPES solution (4-hydroxyethyl piperazine ethanesulfonic acid solution), 10 XDMEM, bovine tail type I collagen at 8:1:1:10:80 to obtain hydrogel (wherein the concentration of NaHCO 3 solution and HEPES solution is 1 mol/L), and placing the hydrogel on ice to prevent solidification; injecting hydrogel into the bottom gel tank 1.1 through a gel sample inlet on the PDMS cover plate, embedding a convex array structure on the PDMS cover plate into the hydrogel in the bottom gel tank 1.1 (injecting excessive hydrogel, filling the whole bottom gel tank 1.1 with the hydrogel, enabling the excessive hydrogel to flow out of the gel sample outlet, avoiding the existence of bubbles in the formed gel bracket 3), and then incubating at 37 ℃ for 20-30min (the incubation time in the embodiment is 30 min) to solidify the hydrogel, so as to obtain the gel bracket 3 with the micro-concave array structure on the surface;
(6) Constructing a lumen channel: horizontally extracting the two lumen columns from the peripheral pouring liquid storage tank by using forceps so as to form two parallel first lumens 1.2 and second lumens 1.3 (because PDMS has ductility and is convenient for extracting the lumen columns, the aperture of the part of the first lumen 1.2 and the second lumen 1.3 which are positioned in the gel support 3 is different from the aperture of the part positioned in the chip main body 1), wherein the first pouring liquid storage tank 1.4 and the third pouring liquid storage tank 1.6 which are symmetrically arranged relative to the concave part are communicated through the first lumen 1.2, the second pouring liquid storage tank 1.5 and the fourth pouring liquid storage tank 1.7 which are symmetrically arranged relative to the concave part are communicated through the second lumen 1.3, and the first lumen 1.2 and the second lumen 1.3 penetrate through the gel support 3;
(7) Change PDMS apron for curb plate 2: and slowly taking down the PDMS cover plate, and then embedding and assembling with the upper side plate 2, so that the upper side plate 2 is embedded in the concave part of the chip main body 1, and the top liquid storage pool 2.1 and the bottom gel pool 1.1 are mutually communicated.
Example 2
The pig endometrium microfluidic chip constructed in the embodiment 1 is adopted, the surface culture of the gel bracket 3 of the pig endometrium microfluidic chip is injected with pig endometrium epithelial cells, the lumen channel culture of the first lumen 1.2 and the second lumen 1.3 is injected with pig endothelial cells, and the specific operation is as follows:
(1) Digestion of porcine endothelial cells with pancreatin (0.25% pancreatin solution was added, incubated at 37 ℃ for 2 min), centrifugation, and removal of supernatant; resuspension of pig endothelial cell sediment to a density of 1×10 5～1×107 pieces/mL (density of 1.0×10 5 pieces/mL in this embodiment) by using DMEM complete medium (dmem+10% fetal bovine serum+1% penicillin-streptomycin), obtaining pig endothelial cell suspension, respectively injecting the first lumen 1.2 and the second lumen 1.3 into a part of the lumen channel in the gel support 3, wherein the single-lumen injection amount is 10-30 μl (the single-lumen injection amount is 20 μl in this embodiment), then placing the chip in an incubator in an environment where 37 ℃ and 5% co 2 and 95% air are mixed, turning over four sides for 20-40min (turning over four sides for 30min in this embodiment), absorbing the stock solution every time, and then re-injecting 20 μl of pig endothelial cell suspension into the single lumen, wherein the total injection amount of pig endothelial cell suspension into the first lumen is 80 μl), filling a liquid pool in the first lumen, filling the liquid pool in the first lumen with DMEM 1.37% of the second medium, and completely filling up to 3% of the second medium in the same volume as the first medium channel (37 ℃ and 3.37% of DMEM, and completely filling up to the fourth medium in the incubator in the same volume of the first medium channel and 3.37% of the first medium and 5% of the medium; in the step, in order to prevent the gel bracket 3 from being polluted and dehydrated and dried, and in order to facilitate operation, the PDMS cover plate is embedded and assembled on the concave part of the chip main body 1 in the whole course, and then the operation of injecting the porcine endothelial cell suspension and culturing is carried out;
(2) Digesting pig endometrium epithelial cells with pancreatin (adding 0.25% pancreatin solution, incubating at 37deg.C for 2 min), centrifuging, and discarding supernatant; re-suspending the pig endometrium epithelial cell sediment to 1X 10 4～1×106/mL (re-suspending the pig endometrium epithelial cell sediment to 1.0X 10 5/mL in the embodiment) by using DMEM complete culture medium to obtain pig endometrium epithelial cell suspension, removing the PDMS cover plate of the pig endometrium microfluidic chip after stationary culture in the step (1), replacing the PDMS cover plate with an upper side plate 2, embedding the concave part of the chip main body 1 with the upper side plate 2, mutually penetrating the top liquid storage tank 2.1 and the bottom gel tank 1.1, then injecting the pig endometrium epithelial cell suspension onto the surface of the gel bracket 3, wherein the single injection amount is 100-200 mu L (the single injection amount in the embodiment is 200 mu L), after stationary culture in an incubator in a mixed environment of 5% CO 2 and 95% air at 37℃for 20-40min (stationary culture in this example for 30 min), the suspension of porcine endometrial epithelial cells was aspirated, washed once with PBS buffer (washing off porcine endometrial epithelial cells that did not grow on the surface of the gel support 3 by adherence), and 200. Mu.L of DMEM complete medium was added through the top reservoir 2.1 of the upper plate 2, at which time the DMEM complete medium completely covered the gel support 3, and stationary culture in an incubator in a mixed environment of 37℃5% CO 2 and 95% air (stationary culture time here is not specifically required, depending on the requirements of the subsequent experiments, and stationary culture time here is 24h in this example).
With the increase of the culture time, the pig endometrium epithelial cells proliferate, differentiate and gradually cover the whole surface on the gel bracket 3 to form a compact growing epithelial cell layer; the whole lumen is filled with the porcine endothelial cells as shown in fig. 5-7 (picture data is obtained by observing and photographing with a nikon microscope, wherein fig. 5-6 are culture diagrams of porcine endometrial epithelial cells on the surface of the gel support 3, the tight arrangement among cells can be seen, the growth state is good, and fig. 7 is a culture diagram of porcine endothelial cells in a lumen channel, and the tight arrangement of cells can be seen.
Example 3
Constructing a pig endometrium micro-fluidic chip added with primary matrix cell culture, which comprises the following steps:
1. extraction and culture of porcine primary endometrial stromal cells
Dissecting healthy sow, opening abdominal cavity, finding uterus position, obtaining 5-10cm pig uterine horn tissue, cutting and cleaning longitudinally, cutting the endometrium tissue into about 1mm 3 pieces, washing the endometrium pieces for several times until the supernatant is clear, placing the endometrium pieces in F12 culture medium containing 0.4mg/mL collagenase V and 1.25U/mL disperse enzyme II, incubating at room temperature, determining digestion time and degree according to tissue amount, total time being 40-50min (total time being 50min in this example), adding equal amount of DMEM/F12 complete culture medium to terminate digestion, standing for 2min after forced rotation, precipitating undigested tissue pieces at the bottom of a centrifuge tube, filtering the supernatant through a 100um filter screen and washing with the DMEM/F12 complete culture medium, repeating for 2-3 times (repeating times in this example for 3 times), collecting filtrate, centrifuging at 200g rotation speed for 5min, adding the DMEM/F12 complete culture medium to obtain stromal cells, incubating in a 37 ℃ incubator for 24-48h (incubation time being 48h in this example);
2. cell trypsin digestion to prepare suspension
When the matrix cells in the culture dish are observed to grow to 80-90% by naked eyes, digestion is carried out, the culture solution is sucked, and the culture solution is washed three times by using sterile PBS buffer solution to remove residual serum; adding a proper amount of 0.25% pancreatin solution, and incubating at 37 ℃ for 1-2min (the different cell digestion time varies, and the incubation time is 2min in the embodiment); when observing that the cells shrink obviously under a microscope, adding a complete culture solution containing serum to stop digestion, blowing off adherent cells to the suspension; centrifuging at 1000rpm for 3min, removing supernatant, adding appropriate amount of DMEM/F12 complete culture medium, counting, and diluting to obtain matrix cell suspension with density of 2×10 5/mL;
The porcine endometrial epithelial cells and the porcine endothelial cells are adherent cells, and corresponding porcine endometrial epithelial cell suspensions and porcine endothelial cell suspensions are prepared according to the method of example 2;
3. Pig endometrium micro-fluidic chip containing matrix cells
Taking a matrix cell suspension, centrifuging again to obtain a matrix cell sediment, containing 2X 10 5 matrix cells, fully mixing the matrix cell sediment with a hydrogel (H 2O：NaHCO3 solution: HEPES solution, 10X DMEM and oxtail type I collagen according to the volume ratio of 8:1:1:10:80), and obtaining a mixture containing the hydrogel and the matrix cells (the density of the matrix cells in the mixture containing the hydrogel and the matrix cells is 2X 10 5/mL) after the mixture is fully mixed, constructing a pig endometrium microfluidic chip according to the method of the embodiment 1, wherein a gel scaffold 3 is formed by solidifying the mixture containing the hydrogel and the matrix cells (namely, the hydrogel in the embodiment 1 is replaced by the mixture containing the hydrogel and the matrix cells); then injecting the pig endothelial cell suspension and the pig endometrial epithelial cell suspension respectively for culturing according to the method of the example 2, and standing for 5 days after injecting the pig endometrial epithelial cell suspension and adding the DMEM complete culture medium; the pig endometrium epithelial cells are proliferated, differentiated and gradually cover the whole surface on the gel bracket 3 along with the increase of the culture time, so as to form a compact growing epithelial cell layer; the porcine endothelial cells fill the whole lumen, the matrix cells gradually shuttle, grow well in the gel scaffold 3, and the porcine endometrial epithelial cells and the porcine endothelial cells have the same observation results as those of fig. 5-7, wherein the porcine endometrial epithelial cells are closely arranged and have good growth state; porcine endothelial cells are closely arranged in the lumen channel; FIG. 8 is a graph of stromal cell growth mixed with hydrogels, showing that the cell morphology is longer than the shuttle length of porcine endometrial epithelial cells and porcine endothelial cells;
4. Immunofluorescence staining is carried out on cells in the pig endometrium microfluidic chip after the stationary culture
Pig endometrial epithelial cell staining: taking a pig endometrium micro-fluidic chip subjected to stationary culture for 3 days in the step 3, removing a DMEM complete medium in a top liquid storage tank 2.1, cleaning the surface of a gel bracket 3 by using a PBS buffer solution, injecting 4% paraformaldehyde into the top liquid storage tank 2.1, fixing for 15min, and then cleaning by using the PBS buffer solution; then permeabilized with 0.1% Triton X-100 for 3-5min (permeabilized for 5min in this example), then washed with PBS buffer, poured into the top reservoir 2.1 with CK18 solution diluted 1:200 with 1% BSA, incubated overnight at 4deg.C, then washed with PBS buffer; injecting a secondary antibody solution (selected according to primary antibody correspondence) diluted by 1% BSA (primary antibody) according to 1:200 and a FITC-labeled phalloidin solution diluted by 1:200 by 1% BSA (primary antibody) into a top liquid storage tank 2.1, incubating for 2 hours at room temperature in a dark place, and then adding a PBS buffer solution for washing; then injecting DAPI cell nucleus dye into the top liquid storage pool 2.1, incubating for 10min at room temperature, and then washing by using PBS buffer solution; after the completion of the staining, the three-dimensional image of the pig endometrial epithelial cells is observed by using a fluorescence microscope, as shown in fig. 9, and fig. 9 is a three-dimensional image of the pig endometrial epithelial cells, and the pig endometrial epithelial cells have red fluorescence, so that the pig endometrial epithelial cells grow well in the chip; and the pig endometrium epithelial cells are adhered to the surface of the hydrogel for growth, and the cells are connected with each other to form a single-layer epithelial cell layer.
Pig endothelial cell staining: taking a pig endometrium micro-fluidic chip subjected to stationary culture for 3 days in the step 3, removing a top liquid storage tank 2.1 and a DMEM complete culture medium in each perfusion liquid storage tank, then cleaning the lumen channels of the first lumen 1.2 and the second lumen 1.3 and the surface of the gel support 3 by using PBS buffer solution, adding PBS buffer solution (which is convenient for keeping the surface of the gel support 3 moist in the whole course and preventing dehydration and drying) into the top liquid storage tank 2.1, injecting 4% paraformaldehyde into the lumen channels of the first lumen 1.2 and the second lumen 1.3, fixing for 15min, and then cleaning by using the PBS buffer solution; then, permeabilizing with 0.1% Triton X-100 for 3-5min (permeabilizing for 5min in this example), then washing with PBS buffer, injecting FITC-labeled phalloidin solution diluted 1:200 with 1% BSA into the lumen channels of the first lumen 1.2 and the second lumen 1.3, incubating at room temperature for 2h in the absence of light, and then washing with PBS buffer; injecting DAPI cell nucleus dye into the lumen channels of the first lumen 1.2 and the second lumen 1.3, incubating for 10min at room temperature, and then washing by using PBS buffer solution; after the staining is completed, a fluorescence microscope is used for observation, and as shown in fig. 10, porcine endothelial cells form a single-layer circular channel along the lumen, and the porcine endothelial cells are attached to form a tubular structure.
Example 4
Attachment of trophoblast cells
The method comprises the following specific steps of comparing adhesion rates of pig embryo trophoblast cells (PTC) in pig endometrium chips of different cell types by utilizing a pig endometrium micro-fluidic chip to which Pig Endometrium Epithelial Cells (PEEC) or pig endothelial cells (PIEC) are added or not to explore influence of a maternal cell environment on a pig embryo adhesion process:
1) Diluting Hoechst33342 with deionized water according to the ratio of 1:200 to prepare 5 mug/mL Hoechst33342 reaction solution;
2) Culturing porcine embryo trophoblast cells in a culture dish, digesting when the porcine embryo trophoblast cells (PTC) in the culture dish are grown to 80-90% by adherence, sucking the culture solution, washing with sterile PBS buffer solution for three times, and removing residual serum; adding proper amount of 0.25% pancreatin solution, and incubating at 37 ℃ for 2min (different cell digestion time is different); when observing that the cells shrink obviously under a microscope, adding a complete culture solution containing serum to stop digestion, blowing off adherent cells to the suspension; centrifuging at 1000rpm for 3min, discarding the supernatant, adding 1mL of Hoechst 33342 reaction solution of 5 mug/mL to resuspend cells, and incubating for 1h in an incubator in a mixed environment of 5% CO 2 and 95% air at 37 ℃;
3) Taking out the EP tube, centrifuging at 800rpm for 3min, discarding the supernatant, then re-suspending with PBS buffer, gently blowing for 30s, discarding the supernatant, repeating the steps for 2 times, and then re-suspending with DMEM complete medium to obtain a pig embryo trophoblast (PTC) suspension with fluorescence;
4) Respectively preparing a pig endometrium micro-fluidic chip with pig endometrium epithelial cells and pig endothelial cells, a pig endometrium micro-fluidic chip with pig endometrium epithelial cells and a pig endometrium micro-fluidic chip without cells, wherein the pig endometrium micro-fluidic chip is operated and cultured according to the method of the embodiment 2; in contrast, in the process of preparing the pig endometrium micro-fluidic chip which is used for culturing the pig endothelial cells independently, the pig endometrium epithelial cell suspension is replaced by the same amount of DMEM complete medium; in the process of preparing a pig endometrium micro-fluidic chip which is used for separately culturing pig endometrium epithelial cells, the suspension of the pig endothelial cells is replaced by an equivalent amount of DMEM complete medium; in the process of preparing the pig endometrium micro-fluidic chip without cells, respectively replacing the pig endothelial cell suspension and the pig endometrium epithelial cell suspension with the same amount of DMEM complete culture medium;
5) Respectively sucking the DMEM complete culture medium in the top liquid storage tank 2.1 of the pig endometrium microfluidic chip (comprising the pig endometrium microfluidic chip cultured with pig endometrium epithelial cells and pig endothelial cells, the pig endometrium microfluidic chip independently cultured with pig endometrium epithelial cells and the pig endometrium microfluidic chip not cultured with cells), respectively adding 200uL of the pig embryo trophoblast (PTC) suspension with fluorescence obtained in the step 3) into the top liquid storage tank 2.1, shooting by using a positive fluorescent microscope, and taking four areas for shooting by using each pig endometrium microfluidic chip;
6) Placing the chip into an incubator in a mixed environment of 37 ℃ and 5% CO 2 and 95% air for continuous culture for 3 hours after shooting is completed, taking out the pig endometrium microfluidic chip after finishing, slowly sucking non-adhered fluorescent pig embryo trophoblast (PTC) suspension in a top liquid storage tank 2.1, gently flushing the surface of a gel bracket 3 by using PBS buffer solution, washing the non-adhered pig embryo trophoblast, placing the chip under a forward fluorescent microscope for shooting, and taking four areas for shooting by using each pig endometrium microfluidic chip;
7) Performing data processing on the photographed fluorescent pictures by using imageJ, comparing the adhesion condition of the porcine embryo trophoblast cells under the two photographing, and further measuring and calculating the adhesion rate of the porcine embryo trophoblast cells on a chip; the fluorescence pictures of the pig endometrium micro-fluidic chip (namely the pig endometrium micro-fluidic chip obtained in the embodiment 2) in which the pig endometrium epithelial cells and the pig endothelial cells are cultured are shown in fig. 11-12, wherein fig. 11 is a picture obtained by shooting when the pig embryo trophoblast cells with fluorescence are planted, fig. 12 is a picture obtained by shooting after the non-adhered pig embryo trophoblast cells are washed away after 3 hours of culture, and according to fig. 11-12, it can be seen that more blue fluorescence still exists after the non-adhered pig embryo trophoblast cells are washed away, which indicates that the pig endometrium micro-fluidic chip of the embodiment 2 of the invention can realize and provide the adherent growth of the pig embryo trophoblast cells on hydrogel; fig. 13 is a data result of the adhesion rate of the four pig endometrium microfluidic chips, and it can be seen that when the pig endometrium epithelial cells and the pig endothelial cells are added, adhesion of pig embryo trophoblast cells is more facilitated, and the structure and the function of a real uterus can be more simulated.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (10)
1. The pig endometrium micro-fluidic chip is characterized by comprising a chip main body, wherein the chip main body is of a concave structure formed by protruding at two sides of a concave part in the middle, an upper side plate is embedded in a concave part of the chip main body, a bottom gel pond is arranged on the concave part of the chip main body, a top liquid storage pond penetrating through the upper side plate is arranged on the upper side plate, the top liquid storage pond and the bottom gel pond are communicated with each other, a gel bracket is filled in the bottom gel pond, and a bionic uterine gland structure is arranged on the surface of the gel bracket;
The convex part of the chip main body is provided with a plurality of groups of pouring liquid storage tank groups, each group of pouring liquid storage tank group consists of two pouring liquid storage tanks which are respectively arranged on the convex part of the chip main body, and the two pouring liquid storage tanks are symmetrically arranged relative to the concave part;
wherein, two perfusion liquid storage tanks which are symmetrically arranged relative to the concave part are communicated through a pipe cavity, and the pipe cavity penetrates through the gel bracket.
2. The pig endometrium microfluidic chip according to claim 1, wherein the gel scaffold is formed by curing a hydrogel or a mixture containing a hydrogel;
The mixture containing the hydrogel also contains matrix cells and/or immune cells;
In the hydrogel-containing mixture, the density of cells was 2×10 5～2×107 cells/mL;
the bionic uterus gland structure is a micro-concave array structure;
The bionic uterine gland structure occupies 40% or more of the surface area of the gel scaffold.
3. The pig endometrium microfluidic chip according to claim 1 or 2, wherein each perfusion liquid storage tank has a cuboid structure, the height is 3-6mm, the length is 5-10mm, and the width is 2-5mm;
the bottom gel tank is of a regular hexagonal prism structure, the side length is 4-8mm, and the height is 1-3mm.
4. The pig endometrium microfluidic chip according to claim 1, wherein a plurality of tube cavities are arranged in parallel, and the length of each tube cavity is 10-20mm;
The aperture of the part of each tube cavity positioned in the gel bracket is 0.4-1mm, and the aperture of the part of each tube cavity positioned in the chip main body is 0.8-1.5mm;
The height of the gel support is 2-4mm.
5. The pig endometrium microfluidic chip according to claim 1, wherein the top liquid storage tank is of a regular hexagonal prism structure, and the cross sections of the top liquid storage tank and the bottom gel tank are the same in size and 2-4mm in height.
6. A method of preparing a pig endometrium microfluidic chip according to any one of claims 1 to 5, comprising the steps of:
S1, designing a pig endometrium micro-fluidic chip module structure, preparing a mould by using a3D printing instrument and a photoetching method, pouring a mixture of polydimethylsiloxane prepolymer and a curing agent, solidifying, demolding and cutting to obtain a chip main body, an upper side plate, a tube cavity column and a cover plate with a convex array structure, wherein the chip main body is of a concave structure formed by protruding at two sides of a concave part in the middle part, a bottom gel pool is formed in the concave part of the chip main body, a top liquid storage pool penetrating through the upper side plate is formed in the upper side plate, a plurality of groups of pouring liquid storage pool groups are formed in the protruding part of the chip main body, each group of pouring liquid storage pool group consists of two pouring liquid storage pools respectively formed in the protruding part of the chip main body, and the two pouring liquid storage pools are symmetrically arranged relative to the concave part;
S2, perforating the side wall of each pouring liquid storage tank to enable the pouring liquid storage tanks to be communicated with a bottom gel tank, injecting hydrogel or a mixture containing the hydrogel into the bottom gel tank, forming a gel bracket with a bionic uterine gland structure on the surface in the bottom gel tank by utilizing a lumen column and a cover plate with a convex array structure, forming a plurality of lumens simultaneously, and communicating two pouring liquid storage tanks symmetrically arranged relative to a concave part by the lumens, wherein the lumens penetrate through the gel bracket, then assembling with an upper side plate, enabling the concave part of a chip main body to be embedded with the upper side plate, and enabling the top liquid storage tank and the bottom gel tank to be mutually communicated.
7. The method according to claim 6, wherein in step S1, the weight ratio of the polydimethylsiloxane prepolymer and the curing agent is (8-12): 1.
8. The method according to claim 6 or 7, wherein the preparation method of the hydrogel in step S2 comprises: h 2O：NaHCO3 solution: HEPES solution: 10 XDMEM: hydrogel monomer = 8:1:1:10: mixing at a volume ratio of 80-180;
The hydrogel monomer is selected from one or more of bovine tail type I collagen, rat tail type I collagen and gelatin.
9. Use of a pig endometrium microfluidic chip according to any one of claims 1-5, characterized in that the use comprises culturing pig endometrium epithelial cells and pig endothelial cells and in vitro mimicking the cellular composition of pig uterus and in vitro mimicking the three-dimensional structure of pig uterus.
10. A method of using the pig endometrium microfluidic chip according to any one of claims 1 to 5, comprising the steps of:
(a) Injecting the pig endothelial cell suspension into each lumen of the chip, turning over four sides, standing for 20-40min respectively, enabling pig endothelial cells to closely adhere to and grow in a lumen channel of each lumen, then adding a complete culture medium into each perfusion liquid storage tank respectively, and standing for 20-30min;
Wherein the density of the porcine endothelial cell suspension is 1X 10 5～1×107/mL, and the dosage of single-lumen injection is 10-30 mu L;
(b) Injecting the pig endometrium epithelial cell suspension onto the gel bracket, standing and culturing for 20-40min, sucking the pig endometrium epithelial cell suspension, cleaning with PBS buffer solution, adding complete culture medium into the top liquid storage tank, and standing and culturing;
Wherein the density of the pig endometrium epithelial cell suspension is 1×10 4～1×106/mL, and the dosage of single injection is 100-200 μl.
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