Testis organ chip based on microfluidics as well as preparation method and application of testis organ chip

Abstract

The invention relates to a testis organ chip based on microfluidics as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The testis organ chip comprises two micro-channel units, a membrane layer structure, spermatogonial stem cells, testis supporting cells and testis interstitial cells, the spermatogonial stem cells and the supporting cells are located in one of the micro-channel units to form a spermatogenic layer; the testicular interstitial cells are located in the other micro-channel unit to form a nutrition layer; the nutrition layer is used for conveying nutrient substances to the spermatogenesis layer through the membrane layer structure. The testis organ chip based on microfluidics has a bionic 3D structure and can simulate a testis organ structure and carry out reproductive pharmacology / reproductive toxicology detection.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a microfluidics-based testis organ chip and a preparation method and application thereof. Background Art

[0002] At present, there are a variety of research platforms for exploring the effects of reproductive damage factors on male fertility, mainly including three categories: animal models, testicular cell culture, and testicular organoid culture. However, the above research platforms have defects in studying reproductive pharmacology and reproductive toxicology.

[0003] Animal models: Currently, animal models mainly include mice, rats, cows, etc. The research process of this model is long, expensive, laborious, and has poor reproducibility. Moreover, although the sex determination and testicular function systems of mice and humans are generally similar, they are not completely conservative, and many genetic diseases caused by single gene defects cannot be successfully modeled in mice.

[0004] Testicular cell culture model: There is currently no reliable testicular cell line that can recapitulate all types of testicular cells in vivo. However, 2D culture of primary testicular cells in serum-containing medium leads to a loss of their normal characteristics, and the gene expression pattern rapidly diverges from that of normal testes.

[0005] Testicular organoid culture model: This method is to extract primary cells or stem cells from mouse or human testicles. First, various testicular cells are obtained, and 3D culture technology is used to make various cells self-assemble to produce spherical testicular organoids. Through intercellular communication of various cells, testicular cells maintain their characteristics. It can be used as a useful and powerful platform for exploring normal testicular development and related pathologies. However, the testicles have a continuous circulatory system. Reproductive drugs and reproductive toxins will pass through the capillaries and the blood-testis barrier to enter and exit the testicles, which has an important impact on spermatogenesis in the testicles. The culture environment of testicular organoids cannot simulate the effects of reproductive drugs / toxins on spermatogenesis in the circulatory system. In short, the current lack of effective research models for the pathogenesis of male infertility limits the development of new technologies in reproductive pharmacology and reproductive toxicology. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a testis organ chip based on microfluidics and a preparation method and application thereof.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] In the first aspect, the present invention provides a testicular organ chip based on microfluidics, wherein the testicular organ chip comprises two microchannel units, a membrane layer structure, spermatogonial stem cells (Spermatogonia Stem Cells, SSC), testicular supporting cells (Sertoli Cells, SC) and testicular interstitial cells (Leydig Cells, LC); the microchannel unit comprises an inlet microchannel, a culture microchannel and an outlet microchannel; the culture microchannels in the two microchannel units are respectively located on both sides of the membrane layer structure to form a sandwich structure; the bottom of the culture microchannel is respectively connected to the two sides of the membrane layer structure; fibronectin and type IV collagen are respectively attached to both sides of the membrane layer structure; the spermatogonial stem cells and supporting cells are located in the culture microchannel of one of the microchannel units to form a spermatogenic layer; the testicular interstitial cells are located in the culture microchannel of the other microchannel unit to form a nutrient layer; the nutrient layer transports nutrients to the spermatogenic layer through the membrane layer structure.

[0009] The present invention constructs a testicular organ chip based on microfluidics, which has a bionic 3D structure that can simulate the function of human testicular organoids. The chip of the present invention cultured spermatogonial stem cells, supporting cells and interstitial cells separately through a membrane layer structure with a certain degree of permeability, but it can ensure that the interstitial cells provide the required nutrients for the spermatogenic layer as a nutrient layer. This separate and independent culture method also facilitates the subsequent separation of spermatogonial stem cells, supporting cells and interstitial cells. At the same time, fibronectin and type IV collagen are attached to both sides of the membrane layer structure to improve the adhesion of cells on both sides of the membrane layer structure, ensuring that the cells located in the lower layer will not have the risk of falling and will always be attached to the membrane layer structure.

[0010] As a preferred embodiment of the first aspect, the width of the inlet microchannel and the outlet microchannel is 1 mm; the length of the culture microchannel is 12 mm, the width is 7 mm, and the depth is 0.9 mm; the length of the membrane layer structure is 20 mm, the width is 7 mm, and the thickness is 0.5 mm.

[0011] As a preferred embodiment of the first aspect, the spermatogonial stem cells of the spermatogenic layer, the testicular supporting cells and the testicular interstitial cells of the nutrient layer are respectively adhered to both sides of the membrane layer structure. The cells adhere to both sides of the membrane layer structure to simulate the testicular seminiferous epithelial structure in terms of cell composition and biological function, and provide the spermatogenic layer with the necessary components for the growth of spermatogonial stem cells through the interstitial cells.

[0012] As a preferred implementation of the first aspect, the membrane layer structure is a carbonate polyester filter membrane with a thickness of 0.2 mm and a filtration pore size of 0.4 μm.

[0013] The present invention adopts carbonate polyester filter membrane as the membrane layer structure, so that it has better permeability and is more conducive to the transportation of nutrients.

[0014] As a preferred embodiment of the first aspect, it further comprises an inlet microchamber and an outlet microchamber, wherein the radius of the inlet microchamber and the outlet microchamber is 1 mm respectively; and the inlet microchamber and the outlet microchamber are connected to the culture medium container.

[0015] As a preferred embodiment of the first aspect, a detection function module is also included. In the present invention, by detecting the difference in the composition of the input cell culture fluid and the output culture fluid in the chip channel (difference in nutrient solution composition, difference in cell secretion composition, cell proliferation state detection, etc.), a detection function module is formed, and the self-assembly of cells in the testicular organ chip, the expression of key functional genes of three functional cells, and when reproductive pharmacological / toxicological factors are introduced into the microfluidic testicular organ chip culture environment, the changes in the main functional indicators of the organ chip can be dynamically evaluated to evaluate the effects of pharmacological / toxicological factors on spermatogenesis.

[0016] In a second aspect, the present invention provides a method for preparing a testis organ chip, comprising the following steps:

[0017] Step 1: Use polydimethylsiloxane to heat and cure in a mold to obtain an overall structure in which two microchannel units are connected together;

[0018] Cut the culture microchannel into two parts, upper and lower, to obtain two independent microchannel units;

[0019] Punching holes at the inlet microchannel and outlet microchannel positions of the microchannel unit to obtain the inlet microchannel and outlet microchannel of the microchannel unit;

[0020] Step 2: Prepare the polyester carbonate filter membrane;

[0021] Step 3: sandwiching the culture microchannels of the two microchannel units of the testis organ chip in step 1 with a carbonate polyester filter membrane to form a sandwich structure and hot-pressing and bonding them to obtain a testis organ chip structure;

[0022] Step 4: Spermatogonial stem cells, testicular supporting cells and testicular interstitial cells are introduced into the culture microchannel through the inlet microchannel and then cultured.

[0023] As a preferred implementation of the second aspect, it also includes adding a mixed coating of fibronectin and / or collagen IV on both sides of the membrane structure.

[0024] The present invention adds fibronectin and / or collagen IV mixed coatings on both sides of the membrane structure respectively. These components form the basal layer of the seminiferous tubules, which can improve the adhesion of cells in culture and enhance the adhesion of cells on the membrane structure.

[0025] As a preferred implementation of the second aspect, the step of cell input in step 4 is specifically as follows:

[0026] Step 1: implanting supporting cells into a culture microchannel of one of the microchannel units and allowing them to remain stationary so that they adhere to one side of the membrane structure;

[0027] Step 2: implanting the Leydig cells into the culture microchannel of another microchannel unit and allowing them to remain stationary so that they adhere to the other side of the membrane structure;

[0028] Step 3: Spermatogonial stem cells are implanted into the culture microchannel of the microchannel unit to which supporting cells are attached.

[0029] The cell planting sequence in the chip of the present invention is specifically designed: according to the histological structure and physiological function of the testicular seminiferous tubules, the testicular supporting cells are first planted to provide conditions for cell colonization for spermatogonial stem cells, and the secretory products of the testicular supporting cells provide conditions for the good proliferation of spermatogonial stem cells. The secretory products of interstitial cells are planted on the other side of the filter membrane to provide nutritional support for spermatogonial stem cells and testicular supporting cells, simulating the cell composition and physiological function outside the testicular seminiferous tubules.

[0030] In a third aspect, the present invention provides the use of the microfluidics-based testis organ chip in drug screening.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The testis organ chip of the present invention has a unique membrane structure, which can promote early cell-to-cell adhesion and cell-to-membrane adhesion, and promote the completion of cell self-assembly and rearrangement to realize the construction of the basal chamber.

[0033] Animal models can be used for pharmacology / toxicology research, but the research process is long, expensive and laborious, and the repeatability is poor. Exploring the effects of pharmacology / toxicology factors on the male reproductive system is too general and difficult to study in detail. The testicular organ chip of the present invention has the advantages of strong operability, strong sustainability and low cost.

[0034] Compared with cell culture models and organoid models, the testicular organ chip of the present invention is closer to the histology and physiology of the male reproductive system, and has inherent advantages in terms of research platform. In addition, the microfluidic organ chip can effectively control the time / dose and other conditions of the testicular organoid tissue in the organ chip affected by pharmacological / toxicological factors, and can more efficiently study the effects of various factors on the male reproductive system, especially spermatogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1Schematic diagram of the testis organ chip of the present invention (A. Schematic diagram of the overall structure of the testis organ chip, in which the basement membrane is a membrane structure; B. Schematic diagram of the three-dimensional structure of the testis organ chip; C. Front and rear views of the chip running in a microfluidic state, in which the arrows show the direction of liquid flow in the chip channel; D. Schematic diagram of the nutrient layer organ chip channel for planting testicular interstitial cells, in which the nuclei of the testicular interstitial cells are stained with Hoechst33342);

[0036] Figure 2 Schematic diagram of the fluorescence detection results of the adhesion between testicular functional cells and membrane layer structures (A. Schematic diagram of testicular functional cell culture and verification; B. Schematic diagram of the adhesion between the membrane layer structure and cells after the addition of fibronectin and collagen IV, in which spermatogonial stem cells are SSCs, testicular supporting cells are SCs and testicular interstitial cells are LCs);

[0037] Figure 3 Schematic diagram of the test results of cell function and membrane layer structure permeability in the testis organ chip (A. Schematic diagram of the immunofluorescence staining results of ZO-1 antibody; B. Schematic diagram of the secretion level of inhibin B detected in the spermatogenic layer; C. Schematic diagram of the testosterone secretion level detection results in the nutrient layer; D. Schematic diagram of testosterone detected in the spermatogenic layer);

[0038] Figure 4 Schematic diagram of the testicular organ chip's proliferation function test results of various testicular cells (A. Schematic diagram of the gradual formation of spermatogonial stem cell colonies; B. Schematic diagram of the 3D reconstruction of the organ structure on the testicular chip captured by a confocal microscope; C. Schematic diagram of the testicular stem cell proliferation level test results in the spermatogenic layer);

[0039] Figure 5 Schematic diagram of the proliferation and differentiation function detection of spermatogonial stem cells in the testis organ chip and its potential for pharmacological and toxicological screening (A. Schematic diagram of the immunofluorescence staining results of the spermatogonial stem cell marker UCHL1; B. Schematic diagram of the immunofluorescence staining results of the spermatogonial differentiation marker C-KIT; C. Schematic diagram of the protein blotting results of the spermatogonial stem cell proliferation marker UCHL1 and the spermatogonial stem cell differentiation marker C-KIT on the 0th and 28th days of chip culture; D. Statistical graph of the protein blotting results of the spermatogonial stem cell proliferation marker UCHL1 and the spermatogonial stem cell differentiation marker C-KIT on the 0th and 28th days of chip culture; E. Schematic diagram of the results of the proliferation level detection of spermatogonial stem cells in the testis organ chip cultured in the culture medium containing PBS / busulfan / hydrogen peroxide). DETAILED DESCRIPTION

[0040] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0041] Example 1

[0042] This embodiment 1 provides a testis organ chip based on microfluidics, and the overall structure of the testis organ chip is as follows Figure 1 As shown in A, it includes two microchannel units, a membrane layer structure, spermatogonial stem cells, testicular supporting cells and testicular interstitial cells; the microchannel unit includes an inlet microchannel, a culture microchannel and an outlet microchannel; the culture microchannels in the two microchannel units are respectively located on both sides of the membrane layer structure to form a sandwich structure; the bottoms of the culture microchannels are respectively connected to the two sides of the membrane layer structure; fibronectin and type IV collagen are respectively attached to both sides of the membrane layer structure; the spermatogonial stem cells and testicular supporting cells are located in the culture microchannel of one of the microchannel units to form a spermatogenic layer; the testicular interstitial cells are located in the culture microchannel of the other microchannel unit to form a nutrient layer; the nutrient layer transports nutrients to the spermatogenic layer through the membrane layer structure.

[0043] Furthermore, the width of the inlet microchannel and the outlet microchannel is 1 mm; the length of the culture microchannel is 12 mm, the width is 7 mm, and the depth is 0.9 mm; the length of the membrane structure is 20 mm, the width is 7 mm, and the thickness is 0.5 mm.

[0044] Furthermore, a fibronectin / collagen IV mixed coating is attached to both sides of the membrane structure.

[0045] Furthermore, the spermatogonial stem cells of the spermatogenic layer, the testicular supporting cells and the testicular interstitial cells of the nutrient layer are respectively adhered to the two sides of the membrane structure.

[0046] Furthermore, the membrane layer structure is a carbonate polyester filter membrane with a filtration pore size of 0.4 μm.

[0047] Furthermore, the overall structure of the testis organ chip is as follows Figure 1 As shown in B, it also includes an inlet microchamber and an outlet microchamber, and the diameters of the inlet microchamber and the outlet microchamber are 1 mm respectively; the inlet microchamber and the outlet microchamber are connected to the culture liquid container.

[0048] Furthermore, the overall structure of the testis organ chip is as follows Figure 1 As shown in C, it also includes a detection function module.

[0049] Example 2

[0050] This embodiment provides a method for preparing a testis organ chip based on microfluidics, comprising the following steps:

[0051] Step 1: Use AutoCAD to draw Figure 1The diagram of the testis organ chip shown in A includes: two microchannel units, each of which includes an inlet microchannel, a culture microchannel and an outlet microchannel.

[0052] Step 2: The pattern described in step 1 is made into a mask plate, and the corresponding testis organ chip mold is obtained by photolithography technology.

[0053] Step 3: Use polydimethylsiloxane (PDMS) to heat and cure in the mold to obtain an overall structure where two microchannel units are connected together;

[0054] Cut the culture microchannel into two parts, upper and lower, to obtain two independent microchannel units;

[0055] Punching holes is performed at the inlet microchannel and outlet microchannel positions of the microchannel unit to obtain the inlet microchannel and outlet microchannel of the microchannel unit.

[0056] Step 4: Prepare a carbonate polyester filter membrane, cut the filter membrane into a size of 12 mm in length and 7 mm in width, and add a mixed coating of fibronectin (50 ug / ul) or / and collagen IV (50 ug / ul) on both sides of the filter membrane;

[0057] Step 5: sandwiching the carbonate polyester filter membrane between the culture microchannels of the two microchannel units of the testis organ chip in step 3 to form a sandwich structure and hot-pressing and bonding them to obtain a testis organ chip structure;

[0058] Step 6: Spermatogonial stem cells, testicular supporting cells and testicular interstitial cells are introduced into the culture microchannel and then connected to the culture medium, and cultured for 28 days to obtain a testicular organ chip;

[0059] 6-1: Spermatogonial stem cells, supporting cells and interstitial cells were prepared into 5×10 5 / ml cell suspension;

[0060] 6-2: The cell suspension of the testicular supporting cells is implanted into the culture microchannel of one of the microchannel units through the microfluidic system and left to stand for 3 days to allow the cells to adhere to one side of the membrane structure;

[0061] 6-3: On the 4th day, the cell suspension of Leydig cells was implanted into the culture microchannel of another microchannel unit through the microfluidic system and left to rest for 3 days to allow the cells to adhere to the other side of the membrane structure;

[0062] 6-4: Implanting the cell suspension of spermatogonial stem cells into the culture microchannel of the microchannel unit to which the testicular supporting cells are attached.

[0063] The present invention is designed according to the histological structure and physiological function of the testicular seminiferous tubules through a specific cell implantation sequence. First, the supporting cells are implanted to provide conditions for the colonization of spermatogonial stem cells, and the secretory products of the supporting cells provide conditions for the good proliferation of spermatogonial stem cells. The secretory products of interstitial cells are implanted on the other side of the filter membrane to provide nutritional support for spermatogonial stem cells / supporting cells, simulating the cell composition and physiological function outside the testicular seminiferous tubules.

[0064] Test Example 1 Adhesion test of the membrane structure of the testis organ chip

[0065] Since cells at different positions on the chip are subject to different gravity, cells at the lower layer will be subject to stronger gravity, and at the same time, the fluid dynamics of the microfluid will affect the adhesion of the cells, causing the risk of the cells at the lower layer falling from the membrane layer. Therefore, in order to improve the adhesion of cells on the membrane structure and eliminate the effects of gravity and fluid dynamics, the present invention adds a mixed coating of fibronectin (50ug / ul) or / and collagen IV (50ug / ul) on both sides of the filter membrane. These components simulate the histological structure of the basement membrane of the seminiferous tubules, can help form the basement layer of the seminiferous tubules, and can improve the adhesion of cells in culture.

[0066] The cell adhesion was identified using the specific cell markers UCHL1, SOX9 and CYP11A1 of spermatogonial stem cells, Sertoli cells and Leydig cells.

[0067] The results are as follows Figure 2 As shown in A and B, compared with the BPS group, the affinity between testicular functional cells and the membrane structure was improved by adding a mixed coating of fibronectin and / or collagen IV on the membrane structure of the testis chip.

[0068] Test Example 2: Detection of cell function and membrane structure permeability in testis organ chip

[0069] The cell culture fluid of the nutrient layer and the spermatogenic layer was pushed into the nutrient layer and the spermatogenic layer at an injection speed of 0.1 ml / h by using a syringe pump to provide the nutrients required by the cells, and then the effect of the testicular organ chip of the present invention was tested.

[0070] Testicular Sertoli cells not only serve as the cell substrate for spermatogonial stem cells, but also as a promoter of tight junction formation, participating in paracrine signaling, producing various secretory proteins, and supporting the proliferation and differentiation of spermatogonial stem cells and spermatogonia.

[0071] The tight junctions between Sertoli cells in the spermatogenic layer were evaluated by immunofluorescence staining using ZO-1 antibody (antibody to tight adhesion protein 1). Figure 3 As shown in A, the immunofluorescence staining results of ZO-1 showed that tight junctions were formed between the supporting cells in the spermatogenic layer.

[0072] Inhibin B is a key protein secreted by testicular Sertoli cells. Detection of inhibin B in the spermatogenic layer can verify whether the testicular Sertoli cells in the testicular organ chip have paracrine activity. Figure 3 (B) The gradual increase in inhibin B levels in the culture medium collected from the spermatogenic layer indicates that the Sertoli cells within the testis organ chip have paracrine activity.

[0073] Testosterone is a specific substance secreted by testicular interstitial cells. Detecting the testosterone level in the nutrient layer can verify whether the testicular organ chip can simulate a microenvironment that is conducive to spermatogenesis. Figure 3 (C) Cell culture medium collected from the nutrient layer showed a gradual increase in testosterone levels secreted by Leydig cells, confirming that the organoids in the testicular organ-on-chip model are able to mimic a microenvironment that is conducive to spermatogenesis.

[0074] The permeability of the membrane structure can be verified by detecting testosterone in the spermatogenic layer and inhibin B in the nutrient layer. The results showed that testosterone ( Figure 3 D), indicating the effective permeability of the membrane layer structure, which facilitates the diffusion of paracrine factors across the different layers of the chip.

[0075] The above results indicate that the functions of each cell in the testis organ chip constructed by the present invention are normal.

[0076] Test Example 3: Proliferation function detection of various testicular cells in the testicular organ chip

[0077] 1. Cell staining

[0078] Spermatogonial stem cells were labeled with the red cell membrane dye CM-DIL, and testicular supporting cells were labeled with the green cell membrane dye CM-FDA, so that they were displayed in different colors in the spermatogenesis layer under a fluorescence microscope (spermatogonial stem cells were displayed in red, and testicular supporting cells were displayed in green); testicular interstitial cells were labeled with Hoechst 33342, so that they emitted blue fluorescence under a fluorescence microscope.

[0079] 2. Cultivation and monitoring:

[0080] According to the method in step 4 of Example 2, the three types of cells were implanted into the testis organ chip, and then cultured for 28 days in combination with a microfluidic system, and the proliferation of spermatogonial stem cells was continuously monitored.

[0081] 3. Results:

[0082] Depend on Figure 4A As can be seen, the growth dynamics of spermatogonial stem cells on the testicular organ chip can be effectively observed by fluorescence microscopy. Spermatogonial stem cell colonies gradually formed on days 1, 7, and 14. On the seventh day of culture on the testicular organ chip, the initially dispersed spermatogonial stem cells began to form sparse cell clusters on the testicular supporting cell layer, a phenomenon that marked the beginning of spermatogonial stem cell cluster proliferation. By the fourteenth day, the number and size of these spermatogenic clusters increased, indicating that spermatogonial stem cells successfully proliferated in the tissue environment on the testicular organ chip.

[0083] Depend on Figure 4 B shows that Leydig cells emit blue fluorescence under a fluorescence microscope and are imaged together with Sertoli cells by confocal microscopy to construct a 3D model of the chip structure. This meticulous arrangement shows that while Leydig cells and Sertoli cells adhere tightly to the membrane layer structure, spermatogonial stem cells do not adhere to the membrane layer structure in planar culture, but only to Sertoli cells.

[0084] To verify the intrinsic proliferation properties of spermatogonial stem cells in the microfluidic environment of the testis organ chip, colchicine was administered to Sertoli cells and Leydig cells during the inoculation process to prevent their proliferation. Subsequently, the culture medium containing CCK-8 was collected from the testis chip organ every day. Figure 4 As can be seen from FIG. 3 , the CCK-8 level was found to be gradually increased, further confirming that spermatogonial stem cells successfully proliferated on these chips constructed in the present invention.

[0085] The above results indicate that spermatogonial stem cells have successfully proliferated on the testicular organ chip constructed by the present invention.

[0086] Test Example 4: Proliferation and differentiation function detection of spermatogonial stem cells in the testis organ chip

[0087] Spermatogonial stem cells are the basis of spermatogenesis and can be spermatogonia. In order to verify the differentiation ability of spermatogonial stem cells in the chip, immunofluorescence staining of spermatogonial stem cell marker UCHL1 and spermatogonial stem cell differentiation marker C-KIT was performed on days 7, 14, and 28, respectively.

[0088] The results are as follows Figure 5 As shown in A, although the expression of UCHL1 did not show a significant increase, indicating that spermatogonial stem cells are in a stable maintenance rather than expansion state, the increase in the number of spermatogonia under the microscope indicates that some of these cells have begun to differentiate.

[0089] The results are as follows Figure 5 As shown in B, by day 14, the significant increase in C-KIT expression further indicated that spermatogonial stem cells not only proliferated but also differentiated into spermatocytes on the testis organ chip.

[0090] The results are as follows Figure 5As shown in C, the results of Western blotting also showed that after 28 days of chip culture, spermatogonial stem cells not only maintained their own proliferation, but also showed a trend of differentiating into spermatogonia. ( Figure 5 C, D).

[0091] The above results indicate that the spermatogonial stem cells on the testis organ chip of the present invention have successfully proliferated and differentiated.

[0092] Test Example 5 Potential of Testis Organ Chip for Pharmacology and Toxicology Screening

[0093] Hydrogen peroxide was used to simulate oxidative stress damage, and the chemotherapeutic drug busulfan was used to simulate the toxicological model. Cytotoxicity experiments were performed to test its effects on spermatogonial stem cells and simulate the effects of reproductive toxins on seminiferous tubules. Hydrogen peroxide concentration: 0.1μmol / ml; busulfan concentration: 0.01μmol / ml. The spermatogenic layer of the testicular organ chip was cultured using medium supplemented with hydrogen peroxide or busulfan, and the proliferation level of spermatogonial stem cells was detected.

[0094] The results are as follows Figure 5 As shown in E, compared with the control group, the proliferation of spermatogonial stem cells in the hydrogen peroxide group decreased, indicating oxidative stress interference. The proliferation of spermatogonial stem cells in the busulfan group decreased more significantly. It can be seen that the testicular organ chip constructed by the present invention has significant sensitivity to drug properties or toxicity and can be used for pharmacological and toxicological screening.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A testis organ chip based on microfluidics, characterized in that: The testis organ chip includes two microchannel units, a membrane structure, spermatogonial stem cells, testicular supporting cells and testicular interstitial cells; The microchannel unit includes an inlet microchannel, a culture microchannel and an outlet microchannel; The culture microchannels in the two microchannel units are respectively located on both sides of the membrane layer structure to form a sandwich structure; the bottoms of the culture microchannels are respectively connected to both sides of the membrane layer structure; The membrane structure has a fibronectin / collagen IV mixed coating on both sides; The spermatogonial stem cells and supporting cells are located in a culture microchannel of one of the microchannel units to form a spermatogenesis layer; The Leydig cells are located in a culture microchannel of another microchannel unit to form a nutrient layer; The nutrient layer transports nutrients to the spermatogenic layer through the membrane structure.

2. The testis organ chip according to claim 1, characterized in that: The width of the inlet microchannel and the outlet microchannel are 1 mm respectively; The culture microchannel has a length of 12 mm, a width of 2 mm, and a depth of 0.9 mm; The film layer structure has a length of 20 mm, a width of 7 mm and a thickness of 0.5 mm.

3. The testis organ chip according to claim 1, characterized in that: The spermatogonial stem cells and supporting cells of the spermatogenic layer and the interstitial cells of the nutrient layer are respectively adhered to the two sides of the membrane layer structure.

4. The testis organ chip according to claim 1, characterized in that: The membrane layer structure is a carbonate polyester filter membrane with a thickness of 0.2 mm and a filtration pore size of 0.4 μm.

5. The testis organ chip according to claim 1, characterized in that: It also includes an inlet microchamber and an outlet microchamber, each of which has a diameter of 1 mm; the inlet microchamber and the outlet microchamber are connected to a culture liquid container.

6. The testis organ chip according to claim 1, characterized in that: By detecting the difference in composition between the input cell culture fluid and the output culture fluid in the chip channel, a detection function module is formed.

7. A method for preparing a testis organ chip according to any one of claims 1 to 6, characterized in that: The following steps are involved: Step 1: Use polydimethylsiloxane to heat and cure in a mold to obtain an overall structure in which two microchannel units are connected together; Cut the culture microchannel into two parts, upper and lower, to obtain two independent microchannel units; Punching holes at the inlet microchannel and outlet microchannel positions of the microchannel unit to obtain the inlet microchannel and outlet microchannel of the microchannel unit; Step 2: Prepare the polyester carbonate filter membrane; Step 3: sandwiching the culture microchannels of the two microchannel units of the testis organ chip in step 1 with a carbonate polyester filter membrane to form a sandwich structure and hot-pressing and bonding them to obtain a testis organ chip structure; Step 4: Spermatogonial stem cells, testicular supporting cells and testicular interstitial cells are introduced into the culture microchannel through the inlet microchannel and then cultured.

8. The method for preparing a testis organ chip according to claim 7, characterized in that: The method also includes adding a mixed coating of fibronectin and / or collagen IV on both sides of the membrane structure.

9. The method for preparing a testis organ chip according to claim 8, characterized in that: The steps of cell input in step 4 are as follows: Step 1: implanting supporting cells into a culture microchannel of one of the microchannel units and allowing them to remain stationary so that they adhere to one side of the membrane structure; Step 2: implanting the Leydig cells into the culture microchannel of another microchannel unit and allowing them to remain stationary so that they adhere to the other side of the membrane structure; Step 3: Implanting spermatogonial stem cells into the culture microchannel of the microchannel unit to which supporting cells are attached.

10. Use of the microfluidics-based testis organ chip according to any one of claims 1 to 6 in drug screening.

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