CN116445282B - Microfluidic system and application thereof in constructing bionic organ microenvironment - Google Patents
Microfluidic system and application thereof in constructing bionic organ microenvironment Download PDFInfo
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Abstract
The application relates to the technical field of microfluidic chips, in particular to a microfluidic system and application thereof in constructing a bionic organ microenvironment.
Description
Technical Field
The application relates to the technical field of microfluidic chips, in particular to a microfluidic system and application thereof in constructing a bionic organ microenvironment.
Background
The organ chip aims at culturing cells, tissues and organs in the microfluidic chip, thereby providing an effective research platform for researching and revealing the biological nature of the organ chip. The bionic material can induce cells to realize specific spatial arrangement, and the organ chip can provide fluid conditions, mechanical stimulation, chemical stimulation and the like similar to in-vivo environment, so that more real physiological and even pathological environment can be simulated in vitro. However, the current organ chip has at least the following problems: the construction aspect of the bionic material simulates single biological characteristics, and the aspect of cell culture, the current multi-organ chip is mainly based on simple coupling of single-organ chips, but the co-culture of a plurality of cell lines is less carried out, and the research on cell-cell interaction is ambiguous.
Seminiferous tubules are curved elongated ducts located within the leaves of the male mammalian testes, the initiation site for meiosis and subsequent production of male gametes. Seminiferous tubule epithelium is composed of testis supporting cells (seltoli), located at the outer border of seminiferous tubules, and is capable of nourishing and supporting primordial stem cells (SSC) during early development and causing them to complete meiosis into the lumen of seminiferous tubules. In addition, the tight connection between the Sertoli cells and the corresponding endothelial and interstitial cells form a blood-testosterone barrier together, which is not only beneficial to forming and maintaining microenvironment required by spermatogenesis, but also prevents sperm antigen substances from escaping outside the seminiferous tubules to generate autoimmune reaction. However, in vitro models for researching the function of seminiferous tubules are lacking, parameter control is inaccurate, and standardization and unification are difficult to achieve.
The existing in vitro seminiferous tubule model has low bionic degree; the structure of the blood-testosterone barrier is single, and the barrier function is still different from that in the body; there is no study of differentiation of spermatogonia and interaction of spermatogonia with Sertoli cells.
Disclosure of Invention
The application provides a method for constructing a human seminiferous tubule chip based on a microfluidic device, which has the advantages of simple structure and simple and convenient operation, and can stably generate an in-vitro model of the human seminiferous tubule.
In a first aspect of the present application, there is provided a microfluidic system comprising a microfluidic chip and a cell, the microfluidic chip comprising a first chip body, a second chip body and a porous membrane;
a first channel which does not penetrate through the first chip main body is arranged on one side surface of the first chip main body;
a second channel which does not penetrate through the second chip main body is arranged on one side surface of the second chip main body;
the porous membrane is positioned between one side of the first chip main body provided with the first channel and one side of the second chip main body provided with the second channel;
the first chip main body is provided with a first inlet and a first outlet which penetrate through the first chip main body at corresponding positions of two ends of the first channel;
the first chip main body is provided with a second inlet and a second outlet which penetrate through the first chip main body and are communicated with the two ends of the second channel at corresponding positions of the two ends of the second channel;
at least one part of the first channel and the second channel exchange substances through the porous membrane;
the first inlet and the second inlet are not positioned at the same position.
Preferably, the first channel and the second channel are located at two sides of the porous membrane, and at least a part of the first channel and the second channel are located on the same plane, so as to ensure that the first channel and the second channel can exchange substances.
More preferably, the porous membrane may be any size as long as the porous membrane is secured between portions of the first and second channels that are located on the same plane, and the first and second channels are capable of mass exchange.
Preferably, the materials of the first chip body, the second chip body and the porous membrane are the same or different.
More preferably, the materials of the first chip body and the second chip body are the same or different, and still more preferably, the materials of the first chip body and the second chip body are the same, and even more preferably, the materials of the first chip body, the second chip body and the porous membrane are the same.
Preferably, the material of the first chip body, the second chip body and/or the porous membrane comprises one or more of silicon material, fluorine material, glass quartz material and/or organic high polymer material.
The material is a biocompatible material.
Further preferably, the silicon material includes, but is not limited to, polydimethylsiloxane (PDMS) and the like.
Further preferably, the fluorine material includes, but is not limited to, polytetrafluoroethylene (PTFE) and the like.
Further preferably, the glass-quartz material includes, but is not limited to, quartz and/or glass, and the like.
Further preferably, the organic high molecular polymer material includes, but is not limited to, polymethyl methacrylate (PMMA), polystyrene (PS), polycarbonate (PC), and/or the like.
In one embodiment of the present application, the material of the first chip body, the second chip body and/or the porous membrane is PDMS.
Preferably, the first channel and the second channel may be channels with any shape, so long as at least a part of the first channel and the second channel are located at two sides of the same plane of the porous membrane, and substance exchange can be performed.
Preferably, the first outlet and the second outlet may be at the same location or not.
Preferably, a third channel, a fourth channel or more channels may be further provided on the first chip body and/or the second chip body.
It is further preferred that the different channels in the first chip body may be placed in parallel or in cross, and more preferred that the different channels in the first chip body are independent of each other.
It is further preferred that the different channels in the second chip body may be placed in parallel or in cross, and more preferred that the different channels in the second chip body are independent of each other.
Preferably, the number of channels in the first chip body and the second chip body may be the same or different.
Preferably, the positions and shapes of the channels in the first chip body and the second chip body may be the same or different.
Preferably, when the first chip body and/or the second chip body include a plurality of channels, at least a portion of one channel in the first chip body and a portion of at least one channel in the second chip body are located on both sides of the same plane of the porous membrane.
Preferably, the first chip body and the second chip body may be designed in any shape, such as a rectangular parallelepiped, a cylinder, or the like, as needed.
Preferably, the cell may be any cell, according to the needs of the specific embodiment, further preferably a cell in an organ of any physiological system including nervous system, respiratory system, digestive system, circulatory system, endocrine system, reproductive system, urinary system, immune system and/or motor system.
In one embodiment of the application, the cells are cells of testis origin, including but not limited to, cells of the seminiferous tubules and/or cells within the interstitium of the testis.
Preferably, the testicular seminiferous tubule cells are selected from one or more of a peritesticular myoid cell, a support cell, a germ cell and/or a stem cell, and/or the testicular interstitial cells are selected from one or more of a vascular endothelial cell, a mesenchymal cell, an immune cell, a fibroblast, a nerve cell and a stem cell.
Preferably, the plurality includes two, three or more.
In a second aspect of the present application, a preparation method of the microfluidic system is provided, where the preparation method includes:
1) Designing a micro-fluidic chip template, and manufacturing a micro-fluidic chip die according to the template;
2) Preparing a first chip body and a second chip body;
3) A first channel which does not penetrate through the first chip main body is arranged on one side of the first chip main body; a second channel which does not penetrate through the second chip main body is arranged on one side of the second chip main body;
4) Preparing a porous membrane;
5) Bonding part or all of one side of the first chip body provided with the first channel with the porous membrane, and bonding part or all of one side of the second chip body provided with the second channel with the other side of the porous membrane;
6) Punching holes at corresponding positions at two ends of a first channel on a first chip main body, and arranging a first inlet and a first outlet penetrating through the first chip main body;
punching holes at corresponding positions of two ends of a second channel on the first chip main body, and arranging a second inlet and a second outlet which penetrate through the first chip main body and are communicated with two ends of the second channel;
7) Injecting cells into the first inlet and/or the second inlet;
the first inlet and the second inlet are not positioned at the same position;
at least a portion of the first and second channels may be in mass exchange relationship with the porous membrane.
Preferably, the first channel and the second channel are located at two sides of the porous membrane, and at least a part of the first channel and the second channel are located on the same plane, so as to ensure that the first channel and the second channel can exchange substances.
More preferably, the porous membrane may be any size as long as the porous membrane is ensured between the portions of the first channel and the second channel which are located on the same plane, and the first channel and the second channel can exchange substances.
Preferably, step 1) includes designing a first chip main body template, manufacturing a first chip main body mold, designing a second chip main body template, manufacturing a second chip main body mold, designing a porous membrane template, and preparing a porous membrane mold, wherein the porous membrane mold is a silicon wafer mold with a densely arranged micro-column array arranged on the surface.
Preferably, the preparing of the first chip body and the second chip body in step 2) includes pouring materials for preparing the first chip body and the second chip body onto the templates designed in step 1), respectively, and heating at 80 ℃ overnight.
Preferably, in step 3), the shape, diameter, etc. of the first and second channels may be set as desired for the particular embodiment.
Further preferably, in step 3), a third channel, a fourth channel or more channels may be further provided on the first chip body and/or the second chip body according to the needs of the specific embodiment.
Preferably, step 4) comprises dropping the material for preparing the porous membrane onto a silicon wafer mold having an array of densely arranged micropillars, spin-coating at 3000 rpm, and then placing the silicon wafer mold in an 80 ℃ oven for heating overnight.
Preferably, the bonding in step 5) comprises using a plasma cleaner.
Preferably, any shape and any diameter of the hole puncher can be used for the hole punching in the step 6), so long as the hole puncher can meet the requirements of cell inoculation and waste liquid discharge.
Preferably, the number of channels in step 7) may be one or more.
Preferably, the number, shape and arrangement of the channels are defined as in the first aspect of the present application.
Preferably, the cell may be any cell, according to the needs of the specific embodiment, further preferably a cell in an organ of any physiological system including nervous system, respiratory system, digestive system, circulatory system, endocrine system, reproductive system, urinary system, immune system and/or motor system.
In one embodiment of the application, the cells are cells of testis origin, including but not limited to, cells of the seminiferous tubules and/or cells within the interstitium of the testis.
Preferably, the testicular seminiferous tubule cells are selected from one or more of a peritesticular myoid cell, a support cell, a germ cell and/or a stem cell, and/or the testicular interstitial cells are selected from one or more of a vascular endothelial cell, a mesenchymal cell, an immune cell, a fibroblast, a nerve cell and a stem cell.
Preferably, the first chip body, the second chip body and/or the porous membrane are defined as in the first aspect of the application.
Preferably, the shape of the first chip body, the second chip body and/or the porous membrane is defined as in the first aspect of the application.
In a third aspect of the present application, a microfluidic chip is provided.
Preferably, the definition of the microfluidic chip is the same as in the first aspect of the application.
In a fourth aspect of the present application, a method for manufacturing a microfluidic chip is provided.
Preferably, the preparation method comprises the following steps:
1) Designing a micro-fluidic chip template, and manufacturing a micro-fluidic chip die according to the template;
2) Preparing a first chip body and a second chip body;
3) A first channel which does not penetrate through the first chip main body is arranged on one side of the first chip main body; a second channel which does not penetrate through the second chip main body is arranged on one side of the second chip main body;
4) Preparing a porous membrane;
5) Bonding part or all of one side of the first chip body provided with the first channel with the porous membrane, and bonding part or all of one side of the second chip body provided with the second channel with the other side of the porous membrane;
6) Punching holes at corresponding positions at two ends of a first channel on a first chip main body, and arranging a first inlet and a first outlet penetrating through the first chip main body;
and punching holes at corresponding positions of two ends of the second channel on the first chip main body, and arranging a second inlet and a second outlet which penetrate through the first chip main body and are communicated with two ends of the second channel.
Preferably, the steps are further defined as in the second aspect of the application.
In a fifth aspect of the present application, an application of any one of the above-mentioned microfluidic systems, any one of the above-mentioned microfluidic systems obtained by the above-mentioned construction methods, any one of the above-mentioned microfluidic chips, and any one of the above-mentioned microfluidic chips obtained by the above-mentioned preparation methods in constructing a micro-environment model of a bionic organ is provided.
Preferably, the bionic organ microenvironment model comprises a bionic seminiferous tubule model and/or a bionic testis microenvironment model.
According to a sixth aspect of the present application, a method for constructing a micro-environment model of a bionic organ is provided, where the method includes obtaining a microfluidic system using any one of the above-mentioned microfluidic systems, any one of the above-mentioned construction methods, any one of the above-mentioned microfluidic chips, and any one of the above-mentioned microfluidic chips obtained by the above-mentioned preparation methods.
Preferably, the construction method comprises culturing the cells in a first channel and/or a second channel, the first channel and the second channel being mass exchanged through a porous membrane.
Preferably, the organ comprises an organ from any physiological system including the nervous system, respiratory system, digestive system, circulatory system, endocrine system, reproductive system, urinary system, immune system and/or motor system.
In one embodiment of the application, the organ is from the reproductive system, preferably the organ is a testis of the reproductive system.
In one embodiment of the present application, the construction method includes:
a) Constructing the microfluidic system or the microfluidic chip;
b) Inoculating a first cell into a first channel through a first inlet, and culturing in the first channel;
c) Adding a cell culture medium into the second inlet and flowing into the second channel, wherein the cell culture medium in the second channel enters the first channel through the porous membrane to culture cells, and constructing a bionic organ model; alternatively, a second cell different from the first cell is introduced into the second channel through the second inlet, cultured in the second channel, and a cell culture medium is added through the first inlet and/or the second inlet, wherein the cell culture medium can circulate between the first channel and/or the second channel through the porous membrane, so as to construct the bionic organ microenvironment model.
In one specific embodiment of the application, the bionic organ microenvironment model comprises a bionic seminiferous tubule model and/or a bionic testis microenvironment model, and the construction method comprises the following steps:
1) Injecting a first cell testis seminiferous tubule cell suspension into the first channel through the first inlet;
2) Adding a testicle seminiferous tubule cell culture medium into the second channel through the second inlet, and allowing the testicle seminiferous tubule cell culture medium to enter the first channel through the porous membrane to culture the testicle seminiferous tubule cells, so as to construct a bionic seminiferous tubule model;
alternatively, the construction method includes:
a) Injecting a first cell testis seminiferous tubule cell suspension into the first channel through the first inlet, and adding a testis seminiferous tubule cell culture medium;
b) Injecting a cell suspension of a second cell different from the first cell into the second channel through the second inlet, and adding a cell culture medium of the second cell, thereby constructing a simulated testis microenvironment model;
the second cell comprises an intra-testicular cell.
Preferably, the testicular seminiferous tubule cells are selected from one or more of a peritesticular myoid cell, a support cell, a germ cell and/or a stem cell, and/or the testicular interstitial cells are selected from one or more of a vascular endothelial cell, a mesenchymal cell, an immune cell, a fibroblast, a nerve cell and a stem cell.
In one embodiment of the application, the testicular seminiferous tubule cells are support cells and the testicular interstitial cells are vascular endothelial cells.
In a seventh aspect of the present application, a bionic organ microenvironment model obtained by any one of the above construction methods is provided.
Preferably, the bionic organ microenvironment model comprises a bionic seminiferous tubule model and/or a bionic testis microenvironment model.
According to an eighth aspect of the present application, there is provided a microfluidic system obtained by any one of the above-described methods of construction, a microfluidic chip obtained by any one of the above-described methods of preparation, and an application of any one of the above-described biomimetic organ microenvironment models, where the application includes one or more of physiological structural modeling, molecular mechanism exploration, cellular interactions, drug screening, disease modeling, assisted reproduction, and/or preclinical studies.
Preferably, the use is not a diagnostic and/or therapeutic method of disease.
More preferably, the drug screening is not a therapeutic approach. The drug screening is to detect and evaluate the effect of the drug to determine whether the drug has a therapeutic effect, i.e. the therapeutic effect is not necessarily the only possibility.
The application has at least one of the following beneficial effects:
first: the microfluidic chip prepared by the method comprises a first chip main body, a second chip main body and a porous membrane, is constructed into a sandwich-like structure of 'upper-middle-lower three layers' of 'the first chip main body-the porous membrane-the second chip main body' in a bonding mode, and can be used for generating seminiferous tubules.
Second,: according to the microfluidic chip disclosed by the application, one side of the first chip main body and one side of the second chip main body are provided with one or more channels, and the same or different kinds of cells can be inoculated in different channels of the first chip main body and different channels of the second chip main body respectively, for example, supporting cells can be inoculated in the first channels and cultured until tight connection is formed, so that the supporting cells are attached to the surface of the porous membrane for growth, or the supporting cells are attached to the surface of the porous membrane and the inner wall surface of the first channel for growth. The second channel is inoculated with testicular interstitial cells (e.g., endothelial cells) which are also grown attached to the surface of the porous membrane or attached to the surface of the porous membrane and the inner wall surface of the second channel. During the culturing process, the first channel and the second channel allow the culture mediums with different components to be respectively introduced to simulate different growth microenvironments of different components of the seminiferous tubule structure. The constructed bionic testis microenvironment in vitro model can be used for detecting and characterizing the function of blood-testosterone barriers.
Third,: after the culture is stable, other cells (such as spermatogenic cells) from testis sources can be also inoculated in the first channel, so that the colonization condition and the further development condition of the cells in seminiferous tubules formed by tight connection of support cells can be conveniently observed.
Fourth,: the application can construct a defective human seminiferous tubule model by controlling cell culture conditions and the like, is used as a pathological model for researching seminiferous tubule related diseases, and can be applied to related chemical and drug screening.
The terms "comprising" or "includes" are used in this specification to be open-ended, having the specified components or steps described, and other specified components or steps not materially affected.
All combinations of items to which the term "and/or" is attached "in this description shall be taken to mean that the respective combinations have been individually listed herein. For example, "a and/or B" includes "a", "a and B" and "B". Also for example, "A, B and/or C" include "a", "B", "C", "a and B", "a and C", "B and C" and "a and B and C".
Any type of numbering in the present application, such as the first, second, third, 1), 2), etc. is merely a naming for distinguishing each other, and does not indicate temporal or spatial sequence unless otherwise indicated.
Drawings
Fig. 1: schematic diagram of microfluidic chip, wherein 1-first chip body, 2-porous membrane, 3-second chip body, 4-first channel, 5-second channel, 6-first inlet, 7-first outlet, 8-second inlet, 9-second outlet; wherein a and b are schematic diagrams of microfluidic chips in different directions respectively;
fig. 2: a physical diagram of the microfluidic chip;
fig. 3: schematic diagram of simulated testis microenvironment structure;
fig. 4: a structure diagram of a bionic testis microenvironment simulating in vitro culture for 4-9 days;
fig. 5: a permeability coefficient measurement result;
fig. 6: immunofluorescent staining of blood-testosterone barrier function;
fig. 7: and (5) manufacturing a silicon wafer template design drawing of the porous membrane.
Detailed Description
The technical scheme of the application is further described below with reference to specific embodiments. It should be understood that the particular embodiments described herein are presented by way of example and not limitation. The main features of the present application may be used in various embodiments without departing from the scope of the present application. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of the application and are covered by the claims. The specific embodiment is as follows:
example 1: preparation of microfluidic chip with double chip body
1. Preparing a silicon wafer: according to the designed chip main structure or porous membrane pore diameter (see fig. 7, pore diameter 8 μm, pitch 40 μm per pore), a silicon wafer mold was fabricated by photolithography, and then the silicon wafer was treated with silane (1 h,2h-perfluor ooctayl) silane for 2 or more h by vapor, wherein the mold of the porous membrane 2 was a silicon wafer mold having a densely arranged array of micro-pillars provided on the surface.
2. Preparing a first chip body 1 and a second chip body 3: PDMS (Polydimethylsiloxane) prepolymer (monomer: crosslinker=10:1) was stirred well and placed in a vacuum chamber to eliminate bubbles. The defoamed PDMS was poured onto a silicon wafer and heated in an oven at 80℃overnight. The PDMS is diced to obtain a first chip body 1 and a second chip body 3.
3. A first channel 4 which does not penetrate through the first chip main body 1 is arranged on one side of the first chip main body 1, and a second channel 5 which does not penetrate through the second chip main body 3 is arranged on one side of the second chip main body 3; the channel has an opening on one side of the chip body, i.e. the channel is a structure recessed down on the surface of the first or second chip body, having a depth allowing the passage of liquid and/or allowing the cell to grow on the wall.
4. Preparation of porous film 2: PDMS prepolymer (monomer: crosslinker=15:1) was dropped onto a silicon wafer mold with a densely arranged array of micropillars, spin coated at 3000 rpm, with a porous film thickness slightly less than the height of the micropillars after spin coating, to form a PDMS film with a dense array of holes. The silicon wafer mold was placed in an 80 ℃ oven and heated overnight.
5. And (3) chip bonding: the first chip body 1 and the porous membrane 2 were simultaneously processed on the surface 90 and s using a plasma cleaning machine, and the processed side of the first chip body 1 provided with the first channels 4 and the porous membrane 2 were aligned and bonded, and annealed at 80 ℃ for 3 h to obtain a bond with a single-sided chip body for use.
6. Repeating the above steps, treating the surface 90 s of the bonding compound obtained in the step 5 and the second chip main body 3 by using a plasma cleaning machine, bonding the bonding compound on the other side of the porous film 2 and the side of the second chip main body 3 provided with the second channel 5, and annealing at 80 ℃ for 3 h to obtain the micro-fluidic chip with the double chip main bodies. The middle section of the first channel and the middle section of the second channel are located on the same plane after bonding.
7. The first chip body is perforated at corresponding positions at two ends of the first channel 4, and a first inlet 6 and a first outlet 7 penetrating the first chip body are provided.
8. The first chip main body is perforated at the corresponding positions of the two ends of the second channel 5, and a second inlet 8 and a second outlet 9 which penetrate through the first chip main body and are communicated with the two ends of the second channel 5 are arranged.
In the preparation process, in the microfluidic chip of the double-chip main body after bonding is required to be ensured, the first channel 4 and the second channel 5 can be in any shape, as long as at least one part of the first channel 4 and the second channel 5 are positioned on the same plane, the substance exchange through the porous membrane is ensured, and the first inlet 6 and the second inlet 8 are not positioned on the same position.
A schematic diagram of the prepared microfluidic chip is shown in fig. 1; as shown in fig. 2, it can be seen from fig. 2 that the micro fluidic chip prepared according to one preferred embodiment of the method of the present application, the lighter color liquid enters the upper left section of the first channel 4 from the first inlet 6, flows through the middle section, then flows to the lower right section, the darker color liquid enters the upper right section of the second channel 5 from the second inlet 8, flows through the middle section, then flows to the lower left section, and the middle sections of the first channel 4 and the second channel 5 are located on the same plane.
Example 2: preparation of bionic testis microenvironment model by using microfluidic chip
A bionic seminiferous tubule and a bionic testis microenvironment model were constructed using the microfluidic chip and different cells prepared as described in example 1.
1. Cell preparation: human embryo multipotent stem cells are used for differentiation to Sertoli cells, endothelial cells and the like respectively for standby.
2. Chip pretreatment: the sterilized microfluidic chip of the double-chip body was washed in a plasma washer for 30 s, the whole chip was coated with 1% polylysine solution for 30 minutes, rinsed with PBS for 5 times, and dried for use.
3. Coating protein on the surface of the porous membrane 2: a mixed solution of 100. Mu.g/mL of rat tail collagen type I and matrix collagen (containing 0.5% Matrigel) was injected into the first inlet 6, and the mixture was left standing at 37℃for 2 h. The second inlet 8 is injected with a proper concentration of I-type rat tail collagen solution, and the chip is placed upside down at 37 ℃ for 2h, and is rinsed with PBS for 5 times for standby.
4. Simulation of vascular endothelial cell seeding: human Umbilical Vein Endothelial Cells (HUVEC) were digested with 0.25% trypsin and counted, a suspension of human umbilical vein endothelial cells was introduced through the second inlet 8 at a suitable density, and cultured in an incubator with inverted standing until cells were grown on the porous membrane side, and the non-adherent cells were slightly rinsed off with medium.
5. Simulated seminiferous tubule Sertoli cell seeding: the Sertoli cells were digested with 0.25% trypsin and counted, sertoli cell suspension was introduced through the first inlet 6 at a suitable density, and the cells were grown on the porous membrane side by standing still in an incubator, the non-adherent cells were rinsed off with the medium, and after the inoculated Sertoli cells formed tight junctions, the porous membrane was examined microscopically to form a dense cell layer both up and down, and then the medium was perfused with a perfusion system.
The obtained bionic testis microenvironment is shown in figure 3, and the result shows that the model can generate a bionic 'seminiferous tubule-seminiferous tubule epithelium-porous membrane-vascular endothelial-vascular' structure, namely the bionic testis microenvironment. The bionic seminiferous tubule cultured in vitro for 9 days is shown in figure 4, and the result shows that the model can perform long-term perfusion culture and form a relatively compact cell monolayer; wherein the Sertoli cell monolayer also forms part of a cell mass, possibly with a certain three-dimensional structure.
Example 3: construction of spermatogonial stem cell in vitro development model by using microfluidic chip
A bionic seminiferous tubule and a bionic testis microenvironment model were constructed using the microfluidic chip and different cells prepared as described in example 1.
1. Cell preparation: human embryo pluripotent stem cells are used for differentiation in the directions of Sertoli cells, spermatogonial stem cells and the like respectively for standby.
2. Chip pretreatment: the sterilized microfluidic chip of the double-chip body was washed in a plasma washer for 30 s, the whole chip was coated with 1% polylysine solution for 30 minutes, rinsed with PBS for 5 times, and dried for use.
3. Coating protein on the surface of the porous membrane 2: a mixed solution of 100. Mu.g/mL of rat tail collagen type I and matrix collagen (containing 0.5% Matrigel) was injected into the first inlet 6, and the mixture was left standing at 37℃for 2 h. The second inlet 8 is injected with a proper concentration of I-type rat tail collagen solution, and the chip is placed upside down at 37 ℃ for 2h, and is rinsed with PBS for 5 times for standby.
4. Simulation of vascular endothelial cell seeding: human Umbilical Vein Endothelial Cells (HUVEC) were digested with 0.25% trypsin and counted, a suspension of human umbilical vein endothelial cells was introduced through the second inlet 8 at a suitable density, and cultured in an incubator with inverted standing until cells were grown on the porous membrane side, and the non-adherent cells were slightly rinsed off with medium.
5. Simulated seminiferous tubule Sertoli cell seeding: sertoli cells were digested with 0.25% trypsin and counted, and Sertoli cell suspension was introduced at appropriate density through the first inlet 6 and cultured in an incubator standing until cells had grown on the side of the porous membrane and non-adherent cells were rinsed off with medium.
6. Repeating the above steps, introducing Sertoli cell suspension for multiple times, placing the chip in an incubator, standing vertically, standing upside down, standing sideways, enabling cells to grow on the surface of the porous membrane and the inner wall surface of the first channel in a wall-attached mode to form a hollow three-dimensional cell lumen structure, verifying that the cells form the three-dimensional lumen structure through a microscope, namely, after the seminiferous tubule structure is obtained, filling a culture medium for culture by using a perfusion system.
7. Simulation of seminiferous tubule side primordial stem cell seeding: the primordial stem cells (SSC) were digested with 0.25% trypsin and counted, and a suspension of SSC cells was introduced through the first inlet 6 at a suitable density, and after a certain period of time, the colonisation of primordial stem cells, the interaction with the seltoli cells and their subsequent development were observed.
As a result, it was found that spermatogonial stem cells SSC can be colonized in the above-constructed biomimetic seminiferous tubule structure, and can be used for observing interaction of spermatogonial stem cells SSC with seltoli cells, and subsequent other studies.
Example 4: seminiferous tubule blood-testosterone barrier function detection
1. The intercellular tight junctions (ZO-1) and the seltoli cell marker SOX9 were characterized by immunofluorescence staining to assess the basic structure and possible barrier function of the model.
2. The second inlet 8 was perfused with a medium containing 70kDa FITC-dextran to simulate macromolecular proteins in the blood stream and investigate its permeability barrier function. And (3) carrying out perfusion culture for 24 hours under normal culture conditions, collecting the liquid at the first outlet 7 and the liquid at the second outlet 9, and calculating to obtain the permeability coefficient of the macromolecular substances so as to judge the barrier function of the model.
The results are shown in FIG. 5 and FIG. 6, wherein FIG. 5 shows the results of FITC-dextran permeability coefficient measurement, and the permeability coefficient (P app ) About 7.41×10 -7 cm/s, the permeability coefficient of the supporting cell group is about 1.93×10 -6 cm/s, thereforeThe former was significantly lower by about 61% than the latter, demonstrating that both form a blood-testosterone barrier and that the supporting cell-endothelial cell group has a better barrier function. FIG. 6 shows immunofluorescent staining results for barrier function, where DAPI indicates nuclei of both cells, ZO-1 indicates tight cell-to-cell junctions, and SOX9 is a specific marker for nuclei of Sertoli cells. The results show that dense and tight connection is formed between the simulated vascular endothelial cells and between the simulated seminiferous tubule Sertoli cells, wherein the Sertoli cell layer has partial cell clusters and shows a certain three-dimensional structure.
The preferred embodiments of the present application have been described in detail above, but the present application is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present application within the scope of the technical concept of the present application, and all the simple modifications belong to the protection scope of the present application.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the application can be made without departing from the spirit of the application, which should also be considered as disclosed herein.
Claims (11)
1. An application of a microfluidic system in constructing a bionic organ microenvironment model;
the microfluidic system comprises a microfluidic chip and cells, wherein the microfluidic chip comprises a first chip main body, a second chip main body and a porous membrane;
a first channel which does not penetrate through the first chip main body is arranged on one side surface of the first chip main body,
a second channel which does not penetrate through the second chip main body is arranged on one side surface of the second chip main body,
the porous membrane is positioned between one side of the first chip main body provided with the first channel and one side of the second chip main body provided with the second channel;
the first chip main body is provided with a first inlet and a first outlet which penetrate through the first chip main body at corresponding positions of two ends of the first channel;
the first chip main body is provided with a second inlet and a second outlet which penetrate through the first chip main body and are communicated with the two ends of the second channel at corresponding positions of the two ends of the second channel;
at least one part of the first channel and the second channel exchange substances through the porous membrane;
the first inlet and the second inlet are not positioned at the same position;
the bionic organ microenvironment model is a bionic seminiferous tubule model and/or a bionic testis microenvironment model;
the cells are testis-derived cells, or the cells are multipotent stem cell differentiated support cells or endothelial cells;
the testis-derived cells comprise testis seminiferous tubule cells and/or testis mesenchyme cells;
the testicular seminiferous tubule cells are selected from one or more of testicular peritubular myoid cells, supporting cells, germ cells and/or stem cells,
and/or the number of the groups of groups,
the testicular interstitial cells are selected from one or more of vascular endothelial cells, interstitial cells, immune cells, fibroblasts, nerve cells and stem cells.
2. The use according to claim 1, wherein the materials of the first chip body, the second chip body and the porous membrane are the same or different.
3. The use according to claim 2, wherein the material of the first chip body, the second chip body and/or the porous membrane comprises one or more of a silicon material, a fluorine material, a glass quartz material and/or an organic high molecular polymer material;
the material is a biocompatible material.
4. The construction method of the bionic organ microenvironment model is characterized by comprising the steps of using a microfluidic system;
the microfluidic system comprises a microfluidic chip and cells, wherein the microfluidic chip comprises a first chip main body, a second chip main body and a porous membrane;
a first channel which does not penetrate through the first chip main body is arranged on one side surface of the first chip main body,
a second channel which does not penetrate through the second chip main body is arranged on one side surface of the second chip main body,
the porous membrane is positioned between one side of the first chip main body provided with the first channel and one side of the second chip main body provided with the second channel;
the first chip main body is provided with a first inlet and a first outlet which penetrate through the first chip main body at corresponding positions of two ends of the first channel;
the first chip main body is provided with a second inlet and a second outlet which penetrate through the first chip main body and are communicated with the two ends of the second channel at corresponding positions of the two ends of the second channel;
at least one part of the first channel and the second channel exchange substances through the porous membrane;
the first inlet and the second inlet are not positioned at the same position;
the bionic organ microenvironment model is a bionic seminiferous tubule model and/or a bionic testis microenvironment model;
the cells are testis-derived cells, or the cells are multipotent stem cell differentiated support cells or endothelial cells;
the testis-derived cells comprise testis seminiferous tubule cells and/or testis mesenchyme cells;
the testicular seminiferous tubule cells are selected from one or more of testicular peritubular myoid cells, supporting cells, germ cells and/or stem cells,
and/or the number of the groups of groups,
the testicular interstitial cells are selected from one or more of vascular endothelial cells, interstitial cells, immune cells, fibroblasts, nerve cells and stem cells.
5. The method of claim 4, wherein the first chip body, the second chip body and the porous membrane are made of the same or different materials.
6. The method of claim 5, wherein the material of the first chip body, the second chip body and/or the porous film comprises one or more of a silicon material, a fluorine material, a glass quartz material and/or an organic high molecular polymer material;
the material is a biocompatible material.
7. The method according to claim 4, wherein the method comprises culturing the cells in the first channel and/or the second channel, and exchanging substances in the first channel and the second channel through the porous membrane.
8. The construction method according to claim 7, wherein the construction method comprises the steps of:
1) Injecting a first cell testis seminiferous tubule cell suspension into the first channel through the first inlet;
2) Adding a testicle seminiferous tubule cell culture medium into the second channel through the second inlet, and allowing the testicle seminiferous tubule cell culture medium to enter the first channel through the porous membrane to culture the testicle seminiferous tubule cells, so as to construct a bionic seminiferous tubule model;
alternatively, the construction method includes:
a) Injecting a first cell testis seminiferous tubule cell suspension into the first channel through the first inlet, and adding a testis seminiferous tubule cell culture medium;
b) Injecting a cell suspension of a second cell different from the first cell into the second channel through the second inlet, and adding a cell culture medium of the second cell, thereby constructing a simulated testis microenvironment model;
the second cell comprises an intra-testicular cell.
9. A simulated organ microenvironment model cultured by the method of any one of claims 4-8;
the bionic organ microenvironment model is a bionic seminiferous tubule model and/or a bionic testis microenvironment model.
10. Use of a biomimetic organ microenvironment model according to claim 9, wherein said use comprises one or more of physiological structure modeling, molecular mechanism exploration, cellular interactions, drug screening, disease modeling, assisted reproduction and/or preclinical studies.
11. The use of claim 10, wherein the cellular interactions include interaction of spermatogonial stem cells with supporting cells.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109865540A (en) * | 2019-03-04 | 2019-06-11 | 中国科学院微电子研究所 | Micro-fluidic alveolar chip based on chemical modification |
| CN111971383A (en) * | 2018-05-21 | 2020-11-20 | 深圳华大生命科学研究院 | Bionic intestinal organ chip and preparation method and application thereof |
| CN114507637A (en) * | 2019-03-12 | 2022-05-17 | 南京医科大学 | Complete testicle culture medium and application thereof |
| CN114574540A (en) * | 2020-12-02 | 2022-06-03 | 中国科学院大连化学物理研究所 | Novel coronavirus intestinal infection model construction method based on microfluidic chip |
| CN115595264A (en) * | 2021-07-07 | 2023-01-13 | 上海市第一人民医院（Cn） | Microfluidic freezing culture device suitable for single seminiferous tubule |
| CN115678774A (en) * | 2022-10-11 | 2023-02-03 | 华中科技大学同济医学院附属同济医院 | Ovarian organ chip, manufacturing method and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10087422B2 (en) * | 2011-12-09 | 2018-10-02 | President And Fellows Of Harvard College | Organ chips and uses thereof |
-
2023
- 2023-06-20 CN CN202310727781.3A patent/CN116445282B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111971383A (en) * | 2018-05-21 | 2020-11-20 | 深圳华大生命科学研究院 | Bionic intestinal organ chip and preparation method and application thereof |
| CN109865540A (en) * | 2019-03-04 | 2019-06-11 | 中国科学院微电子研究所 | Micro-fluidic alveolar chip based on chemical modification |
| CN114507637A (en) * | 2019-03-12 | 2022-05-17 | 南京医科大学 | Complete testicle culture medium and application thereof |
| CN114574540A (en) * | 2020-12-02 | 2022-06-03 | 中国科学院大连化学物理研究所 | Novel coronavirus intestinal infection model construction method based on microfluidic chip |
| CN115595264A (en) * | 2021-07-07 | 2023-01-13 | 上海市第一人民医院（Cn） | Microfluidic freezing culture device suitable for single seminiferous tubule |
| CN115678774A (en) * | 2022-10-11 | 2023-02-03 | 华中科技大学同济医学院附属同济医院 | Ovarian organ chip, manufacturing method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Testicular organoid formation is a property of immature somatic cells, which self-assemble and exhibit long-term hormone-responsive endocrine function;Maxwell E Edmonds and Teresa K Woodruff;Biofabrication;第12卷(第4期);1-16 * |
| 小鼠睾丸体外生精小管模型构建及Rho/ROCK信号通路对其重构的影响;孙梦迪等;遵义医科大学学报;第45卷(第2期);147-152 * |
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