A microfluidic-based testis organ chip and a preparation method and application thereof

Abstract

The application relates to a microfluidic-based testis organ chip and a preparation method and application thereof, and belongs to the technical field of biological medicines.The testis organ chip comprises two microchannel 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 microchannel units to form a spermatogenesis layer; the testis interstitial cells are located in the other microchannel unit to form a nutrition layer; and the nutrition layer transports nutrition to the spermatogenesis layer through the membrane layer structure.The microfluidic-based testis organ chip has a bionic 3D structure, can simulate a testis organ structure and can be used for reproductive pharmacology / reproductive toxicology detection.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a microfluidic-based testicular organ-on-a-chip, its preparation method, and its application. Background Technology

[0002] Currently, various research platforms exist for exploring the impact of reproductive damage factors on male fertility, mainly falling into three categories: animal models, testicular cell culture, and testicular organoid culture. However, these platforms all have limitations in studying reproductive pharmacology and reproductive toxicology.

[0003] Animal models: Currently, animal models mainly include mice, rats, and cattle. Research using these models is lengthy, expensive, and laborious, and has poor reproducibility. Furthermore, although sex determination and testicular function systems are generally similar in mice and humans, they are not entirely conserved, and many genetic diseases caused by single-gene defects cannot be successfully modeled in mice.

[0004] Testicular cell culture model: Currently, there is no reliable testicular cell line that can represent all types of testicular cells in vivo. Furthermore, 2D culture of primary testicular cells in serum-containing medium leads to the loss of their normal characteristics, and their gene expression patterns rapidly differentiate from normal testes.

[0005] Testicular organoid culture models: This method involves extracting primary cells or stem cell-based methods from mouse or human testes. Various types of testicular cells are first obtained, and then 3D culture technology is used to allow these cells to self-assemble into spherical testicular organoids. Intercellular communication among the different cell types helps maintain the characteristics of the testicular cells. This can serve as a useful and powerful platform for exploring normal testicular development and related pathologies. However, the testes have a continuous circulatory system. Reproductive drugs and toxins can enter and exit the testes through the capillaries and blood-testis barrier, significantly impacting spermatogenesis. The culture environment of testicular organoids cannot simulate the effects of circulatory drugs / toxins on spermatogenesis. In conclusion, the current lack of effective models for studying 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 this invention is to overcome the shortcomings of the prior art and provide a microfluidic-based testicular organ-on-a-chip, its preparation method and application.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a microfluidic-based testicular organ-on-a-chip, comprising two microchannel units, a membrane structure, spermatogonial stem cells (SSCs), Sertoli cells (SCs), and Leydig cells (LCs). Each microchannel unit includes an inlet microchannel, a culture microchannel, and an outlet microchannel. The culture microchannels in the two microchannel units are located on opposite sides of the membrane structure, forming a sandwich structure. The bottoms of the culture microchannels are connected to both sides of the membrane structure. Fibronectin and type IV collagen are attached to both sides of the membrane structure. The spermatogonial stem cells and Sertoli cells are located in the culture microchannel of one microchannel unit, forming a spermatogenesis layer. The Leydig cells are located in the culture microchannel of the other microchannel unit, forming a nutrient layer. The nutrient layer delivers nutrients to the spermatogenesis layer through the membrane structure.

[0008] This invention constructs a microfluidic-based testicular organochip, which features a biomimetic 3D structure capable of mimicking the function of human testicular organoids. The chip utilizes a permeable membrane structure to separately culture spermatogonial stem cells, Sertoli cells, and testicular interstitial cells, while ensuring that the testicular interstitial cells act as a nutrient layer to provide the necessary nutrients to the spermatogenic layer. This separate, independent culture method also facilitates subsequent separation of spermatogonial stem cells, Sertoli cells, and testicular interstitial cells. Simultaneously, fibronectin and type IV collagen are attached to both sides of the membrane structure, enhancing cell adhesion and ensuring that cells in the lower layer remain attached to the membrane structure without risk of sagging.

[0009] In a preferred embodiment of the first aspect, the inlet microchannel and the outlet microchannel have a width of 1 mm; the culture microchannel has a length of 12 mm, a width of 7 mm, and a depth of 0.9 mm; the membrane structure has a length of 20 mm, a width of 7 mm, and a thickness of 0.5 mm.

[0010] In a preferred embodiment of the first aspect, the spermatogonial stem cells of the spermatogenic layer, the testicular Sertoli cells, and the testicular interstitial cells of the nutrient layer are respectively adhered to both sides of the membrane structure. The cells adhere to both sides of the membrane structure, mimicking the structure of the testicular spermatogenic epithelium in terms of cellular composition and biological function, and providing the necessary components for the growth of spermatogonial stem cells to the spermatogenic layer through the interstitial cells.

[0011] As a preferred embodiment of the first aspect, the membrane structure is a carbonate polyester filter membrane with a thickness of 0.2 mm and a pore size of 0.4 μm.

[0012] This invention uses a carbonated polyester filter membrane as the membrane structure, which improves its permeability and facilitates the transport of nutrients.

[0013] As a preferred embodiment of the first aspect, it further includes an inlet microchamber and an outlet microchamber, the inlet microchamber and the outlet microchamber having a radius of 1 mm respectively; the inlet microchamber and the outlet microchamber are connected to the culture medium container.

[0014] As a preferred embodiment of the first aspect, a detection function module is also included. In this invention, the detection function module is constructed by detecting the differences in composition between the input and output cell culture media in the chip channel (differences in nutrient solution composition, differences in cell secretion components, cell proliferation status detection, etc.). This detection function module can detect in real time the cell self-assembly status in the testicular organ-on-a-chip, the expression of key functional genes in the three types of functional cells, and, when reproductive pharmacological / toxicological factors are introduced into the microfluidic testicular organ-on-a-chip culture environment, can dynamically assess changes in the main functional indicators of the organ-on-a-chip to evaluate the impact of pharmacological / toxicological factors on spermatogenesis.

[0015] Secondly, the present invention provides a method for preparing a testicular organ-on-a-chip, comprising the following steps: Step 1: Polydimethylsiloxane is heated and cured in a mold to obtain an integral structure in which two microchannel units are connected together; The microchannel culture site was cut into two parts, upper and lower, to obtain two independent microchannel units; The inlet and outlet microchannels of the microchannel unit are punched and perforated to obtain the inlet and outlet microchannels of the microchannel unit. Step 2: Prepare the carbonated polyester filter membrane; Step 3: The culture microchannels of the two microchannel units of the testicular organ-on-a-chip from Step 1 are sandwiched with a polyester carbonate filter membrane to form a sandwich structure and then hot-pressed together to obtain the testicular organ-on-a-chip structure. Step 4: Spermatogonial stem cells, testicular Sertoli cells, and testicular interstitial cells are introduced into the culture microchannel through the inlet microchannel and then cultured.

[0016] As a preferred embodiment of the second aspect, it further includes adding a mixed coating of fibronectin and / or collagen IV to both sides of the membrane structure.

[0017] The present invention adds a mixed coating of fibronectin and / or collagen IV to both sides of the membrane structure. These components form the basal layer of seminiferous tubules, which can improve cell adhesion in culture and enhance cell adhesion to the membrane structure.

[0018] As a preferred embodiment of the second aspect, step 4, the cell input step, is specifically as follows: Step 1: Insert the supporting cells into the culture microchannel of one of the microchannel units, let them stand still, and allow them to adhere to one side of the membrane structure; Step 2: Implant testicular interstitial cells into the culture microchannel of another microchannel unit and allow them to remain stationary, allowing them to adhere to the other side of the membrane structure; Step 3: Spermatogonial stem cells are implanted into culture microchannels containing microchannel units with attached supporting cells.

[0019] The cell implantation sequence in the chip of this invention is specifically designed: based on the histological structure and physiological function of the seminiferous tubules of the testis, testicular Sertoli cells are implanted first to provide conditions for the colonization of spermatogonial stem cells, and the secretions of the testicular Sertoli cells provide conditions for the healthy proliferation of spermatogonial stem cells. On the other side of the filter membrane, interstitial cells are implanted to secrete products that provide nutritional support for both spermatogonial stem cells and testicular Sertoli cells, mimicking the cellular composition and physiological function outside the seminiferous tubules of the testis.

[0020] Thirdly, the present invention provides the application of the microfluidic-based testicular organ-on-a-chip in drug screening.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: The testicular organ-on-a-chip of this invention has a unique membrane structure that can promote early cell-to-cell adhesion and cell-to-membrane adhesion, and promote cell self-assembly and rearrangement to achieve the construction of basal chambers.

[0022] Animal models can be used for pharmacological / toxicological studies, but the process is lengthy, expensive, and laborious, with poor reproducibility. Exploring the effects of pharmacological / toxicological factors on the male reproductive system is too general and difficult to study in detail. The testicular organ-on-a-chip of this invention has advantages such as high operability, high sustainability, and low cost.

[0023] This invention, a testicular organ-on-a-chip, offers a more accurate representation of the histology and physiology of the male reproductive system compared to cell culture models and organoid models, providing an inherent advantage in research platform aspects. Furthermore, the microfluidic organ-on-a-chip allows for effective control over the time and dosage conditions of pharmacological / toxicological factors affecting testicular organoid tissue within the organ-on-a-chip, enabling more efficient research into the effects of various factors on the male reproductive system, particularly spermatogenesis. Attached Figure Description

[0024] Figure 1 The following are schematic diagrams of the testicular organ-on-a-chip of the present invention: (A. Schematic diagram of the overall structure of the testicular organ-on-a-chip, wherein the basement membrane is a membrane layer structure; B. Schematic diagram of the three-dimensional structure of the testicular organ-on-a-chip; C. Front and rear views of the chip operating in a microfluidic state, wherein the arrows indicate the direction of liquid flow in the chip channel; D. Schematic diagram of the nutrient layer organ-on-a-chip channel for implanting testicular interstitial cells, wherein the nuclei of the testicular interstitial cells are stained with Hoechst 33342). Figure 2Schematic diagram of fluorescence detection results for adhesion of functional testicular cells to membrane structures (A. Schematic diagram of testicular functional cell culture and verification; B. Schematic diagram of adhesion of membrane structures to cells after the addition of fibronectin and collagen IV, where spermatogonial stem cells are SSC, testicular Sertoli cells are SC and testicular interstitial cells are LC). Figure 3 Schematic diagrams of cell function and membrane structure permeability detection results in testicular organomicroarray (A. Schematic diagram of ZO-1 antibody immunofluorescence staining results; B. Schematic diagram of inhibin B secretion level detection results in spermatogenesis layer; C. Schematic diagram of testosterone secretion level detection results in trophic layer; D. Schematic diagram of testosterone detection in spermatogenesis layer). Figure 4 Schematic diagrams of the results of proliferative function detection of various testicular cells on organomicroarray (A. Schematic diagram of the gradual formation of spermatogonial stem cell colonies; B. Schematic diagram of 3D reconstruction of organ structures on a testicular slice captured by confocal microscopy; C. Schematic diagram of the results of proliferative level detection of spermatogonial stem cells in the spermatogenic layer). Figure 5 This diagram illustrates the potential of testicular organ-on-a-chip (TEA) for detecting the proliferation and differentiation function of spermatogonial stem cells and for pharmacological and toxicological screening. (A. Immunofluorescence staining results of spermatogonial stem cell marker UCHL1; B. Immunofluorescence staining results of spermatogonial cell differentiation marker C-KIT; C. Western blot results of spermatogonial stem cell proliferation marker UCHL1 and spermatogonial stem cell differentiation marker C-KIT on day 0 and day 28 of TEA culture; D. Statistical graph of Western blot results of spermatogonial stem cell proliferation marker UCHL1 and spermatogonial stem cell differentiation marker C-KIT on day 0 and day 28 of TEA culture; E. Schematic diagram of the proliferation level detection results of spermatogonial stem cells in testicular organ-on-a-chip cultured in PBS / busulin / hydrogen peroxide). Detailed Implementation

[0025] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0026] Example 1 This embodiment 1 provides a microfluidic-based testicular organ-on-a-chip, the overall structure of which is as follows: Figure 1As shown in Figure A, the structure includes two microchannel units, a membrane structure, spermatogonial stem cells, testicular Sertoli cells, and testicular interstitial cells. Each microchannel unit includes an inlet microchannel, a culture microchannel, and an outlet microchannel. The culture microchannels in the two microchannel units are located on either side of the membrane structure, forming a sandwich structure. The bottom of each culture microchannel is connected to both sides of the membrane structure. Fibronectin and type IV collagen are attached to both sides of the membrane structure. The spermatogonial stem cells and testicular Sertoli cells are located in the culture microchannel of one microchannel unit, forming a spermatogenesis layer. The testicular interstitial cells are located in the culture microchannel of the other microchannel unit, forming a nutrient layer. The nutrient layer delivers nutrients to the spermatogenesis layer through the membrane structure.

[0027] Furthermore, the inlet microchannel and outlet microchannel have a width of 1 mm; the culture microchannel has a length of 12 mm, a width of 7 mm, and a depth of 0.9 mm; the membrane structure has a length of 20 mm, a width of 7 mm, and a thickness of 0.5 mm.

[0028] Furthermore, the membrane structure is coated with a fibronectin / collagen IV mixed coating on both sides.

[0029] Furthermore, the spermatogonial stem cells of the spermatogenic layer, the testicular Sertoli cells, and the testicular interstitial cells of the nutrient layer are respectively adhered to both sides of the membrane structure.

[0030] Furthermore, the membrane structure is a carbonate polyester filter membrane with a filtration pore size of 0.4 μm.

[0031] Furthermore, the overall structure of the testicular organ-on-a-chip is as follows: Figure 1 As shown in Figure B, it also includes an inlet microchamber and an outlet microchamber, the diameters of which are 1 mm; the inlet microchamber and the outlet microchamber are connected to the culture medium container.

[0032] Furthermore, the overall structure of the testicular organ-on-a-chip is as follows: Figure 1 As shown in C, it also includes a detection function module.

[0033] Example 2 This embodiment provides a method for fabricating a microfluidic-based testicular organ-on-a-chip, comprising the following steps: Step 1: Draw the following using AutoCAD Figure 1 The diagram shown in Figure A is a testicular organ-on-a-chip, which includes two microchannel units, each comprising an inlet microchannel, a culture microchannel, and an outlet microchannel.

[0034] Step 2: The pattern described in Step 1 is made into a mask, and the corresponding testicular organ chip mold is obtained by photolithography.

[0035] Step 3: Use polydimethylsiloxane (PDMS) to heat and cure in a mold to obtain an integral structure in which two microchannel units are connected together; The microchannel culture site was cut into two parts, upper and lower, to obtain two independent microchannel units; The inlet and outlet microchannels of the microchannel unit are punched and perforated to obtain the inlet and outlet microchannels of the microchannel unit.

[0036] Step 4: Prepare a carbonated polyester filter membrane. Cut the filter membrane to a size of 12mm in length and 7mm in width. Add a mixed coating of fibronectin (50ug / ul) and / or collagen IV (50ug / ul) to both sides of the filter membrane. Step 5: The culture microchannels of the two microchannel units of the testicular organ-on-a-chip from Step 3 are sandwiched with a polyester carbonate filter membrane to form a sandwich structure and then hot-pressed together to obtain the testicular organ-on-a-chip structure. Step 6: After introducing spermatogonial stem cells, testicular Sertoli cells, and testicular interstitial cells into the culture microchannel, they are inoculated into culture medium and cultured for 28 days to obtain a testicular organ microarray. 6-1: Spermatogonial stem cells, Sertoli cells, and testicular interstitial cells were prepared into 5×10⁻⁶ cells each. 5 / ml of cell suspension; 6-2: A suspension of testicular supporting cells was implanted into the culture microchannel of one of the microchannel units using a microfluidic system and allowed to stand for 3 days to allow it to adhere to one side of the membrane structure. 6-3: On the 4th day, the cell suspension of testicular interstitial cells was implanted into the culture microchannel of another microchannel unit through a microfluidic system and left to stand for 3 days to allow it to adhere to the other side of the membrane structure. 6-4: Implant a suspension of spermatogonial stem cells into a culture microchannel containing microchannel units with attached testicular supporting cells.

[0037] This invention employs a specific cell implantation sequence designed based on the histological structure and physiological function of the seminiferous tubules of the testis. First, Sertoli cells are implanted to provide conditions for spermatogonial stem cell colonization and, through their secretions, to facilitate healthy proliferation of the spermatogonial stem cells. On the other side of the filter membrane, interstitial cells are implanted, and their secretions provide nutritional support to the spermatogonial stem cells / Sertoli cells, mimicking the cellular composition and physiological function outside the seminiferous tubules of the testis.

[0038] Test Example 1: Adhesion Detection of Testicular Organ-on-a-Chip Membrane Structure Because cells at different locations on the chip experience varying degrees of gravity, with cells in the lower layers experiencing stronger gravity, coupled with the hydrodynamics of microfluidics, cell adhesion is affected, increasing the risk of lower-layer cells detaching from the membrane. Therefore, to improve cell adhesion to the membrane structure and eliminate the effects of gravity and hydrodynamics, this invention adds a mixed coating of fibronectin (50 μg / µl) and / or collagen IV (50 μg / µl) to both sides of the filter membrane. These components mimic the histological structure of the seminiferous tubule basement membrane, aiding in the formation of the seminiferous tubule basal layer and improving cell adhesion during culture.

[0039] Cell adhesion was identified using specific cell markers UCHL1, SOX9, and CYP11A1 from three cell types: spermatogonial stem cells, Sertoli cells, and testicular interstitial cells.

[0040] 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 to the testicular chip membrane structure.

[0041] Test Example 2: Detection of Cell Function and Membrane Structure Permeability in Testicular Organ-on-a-Chip The cell culture medium of the nutrient layer and spermatogenesis layer was injected at a rate of 0.1 ml / h using an injection pump to provide the cells with the necessary nutrients. The effect of the testicular organ chip of the present invention was then tested.

[0042] Sertoli cells in the testes not only serve as cell substrates for spermatogonial stem cells, but also as promoting factors for tight junction formation, participating in paracrine signaling, producing various secretory proteins, and supporting the proliferation and differentiation of spermatogonial stem cells and spermatogonia.

[0043] The tight junctions between Sertoli cells in the spermatogenic layer were assessed using immunofluorescence staining with ZO-1 antibody (cytoplasmic tight adhesion protein 1 antibody). Results are as follows: Figure 3 As shown in Figure A, the immunofluorescence staining results of ZO-1 show that tight junctions are formed between the supporting cells in the spermatogenic layer.

[0044] Inhibin B is a key protein secreted by Sertoli cells in the testis. Detection of inhibin B in the spermatogenic layer can verify whether Sertoli cells within a testis organomicroarray possess paracrine activity. Results are as follows... Figure 3 As shown in Figure B, the gradual increase in the level of inhibin B in the culture medium collected from the spermatogenic layer indicates that the Sertoli cells of the testis within the testis organochip have paracrine activity.

[0045] Testosterone is a specific substance secreted by the interstitial cells of the testis. Detecting testosterone levels in the nutrient layer can verify whether the testicular organ-on-a-chip can simulate a microenvironment conducive to spermatogenesis. Results are as follows... Figure 3 As shown in Figure C, the cell culture medium collected from the nutrient layer showed a gradual increase in the level of testosterone secreted by the interstitial cells of the testis, confirming that the organ model on the testis organ-on-a-chip can simulate a microenvironment conducive to spermatogenesis.

[0046] Detecting testosterone in the spermatogenic layer and inhibin B in the trophoblast layer can verify the permeability of the membrane structure. The results showed that testosterone was detected in the spermatogenic layer. Figure 3 (D) This indicates the effective permeability of the membrane structure, which facilitates diffusion between different layers of the chip via paracrine factors.

[0047] The above results indicate that the cells in the testicular organ-on-a-chip constructed in this invention function normally.

[0048] Test Example 3: Detection of Proliferative Function of Various Testicular Cells Using an Organ-on-a-Chip Microarray 1. Cell staining Spermatogonial stem cells were labeled with the red cell membrane dye CM-DIL, and Sertoli cells were labeled with the green cell membrane dye CM-FDA, so that they appeared in different colors under a fluorescence microscope in the spermatogenic layer (spermatogonial stem cells appeared red, and Sertoli cells appeared green); testicular interstitial cells were labeled with Hoechst 33342 staining, so that they emitted blue fluorescence under a fluorescence microscope.

[0049] 2. Cultivation and monitoring: Following the method in step 4 of Example 2, the three types of cells were implanted into the testicular organochip and cultured for 28 days using a microfluidic system to continuously monitor the proliferation of spermatogonial stem cells.

[0050] 3. Results: Depend on Figure 4 As shown in Figure A, the growth dynamics of spermatogonial stem cells on the testicular organ-on-a-chip can be effectively observed using fluorescence microscopy. Spermatogonial stem cell colonies gradually formed on days 1, 7, and 14. On day 7 of culture on the testicular organ-on-a-chip, the initially dispersed spermatogonial stem cells began to form sparse clusters on the Sertoli cell layer of the testis, marking the beginning of clustered proliferation of spermatogonial stem cells. By day 14, the number and size of these spermatogenic clusters had increased, indicating that spermatogonial stem cells had successfully proliferated in the tissue environment of the testicular organ-on-a-chip.

[0051] Depend on Figure 4 As shown in Figure B, testicular interstitial cells emit blue fluorescence under a fluorescence microscope and are imaged together with testicular Sertoli cells using a confocal microscope to construct a 3D model of the chip structure. This meticulous arrangement indicates that while testicular interstitial cells and testicular Sertoli cells adhere tightly to the membrane structures, spermatogonial stem cells do not adhere to the membrane structures in planar culture, but only to testicular Sertoli cells.

[0052] To verify the intrinsic proliferative characteristics of spermatogonial stem cells in the microfluidic environment of a testicular organ-on-a-chip, colchicine was administered to testicular Sertoli cells and interstitial cells during seeding to inhibit their proliferation. Subsequently, culture medium containing CCK-8 was collected daily from the testicular organ-on-a-chip. Figure 4 As can be seen from C, the CCK-8 level gradually increased, further confirming that spermatogonial stem cells successfully proliferated on these chips constructed in this invention.

[0053] The above results demonstrate that spermatogonial stem cells successfully proliferated on the testicular organ-on-a-chip constructed in this invention.

[0054] Test Example 4: Detection of Proliferation and Differentiation Function of Spermatogonial Stem Cells on Testicular Organ Microarray Spermatogonial stem cells are the basis of spermatogenesis and can be spermatogonia. To verify the differentiation capacity of spermatogonial stem cells in the microarray, immunofluorescence staining was performed on spermatogonial stem cell markers UCHL1 and C-KIT on days 7, 14, and 28, respectively.

[0055] The results are as follows Figure 5 As shown in Figure A, although the expression of UCHL1 did not show a significant increase, indicating that the spermatogonial stem cells were in a stable rather than expanding state, the increase in the number of spermatogonia under the microscope indicated that some of these cells had begun to differentiate.

[0056] The results are as follows Figure 5 As shown in Figure B, the significant increase in C-KIT expression by day 14 further indicates that spermatogonial stem cells not only proliferate but also differentiate into spermatocytes on the testicular organelle chip.

[0057] The results are as follows Figure 5 As shown in Figure C, the results of Western blotting also indicated that after 28 days of microarray culture, spermatogonial stem cells not only maintained their own proliferation but also showed a tendency to differentiate into spermatogonia. Figure 5 C, D).

[0058] The above results demonstrate that spermatogonial stem cells on the testicular organ chip of this invention have successfully proliferated and differentiated.

[0059] Test Example 5: The Potential of Testicular Organ-on-a-Chip for Pharmacological and Toxicological Screening Hydrogen peroxide was used to simulate oxidative stress damage, and the chemotherapy drug busulfan was used to simulate a toxicological model. Cytotoxicity experiments were conducted to test their effects on spermatogonial stem cells and the effects of simulated reproductive toxins on seminiferous tubules. Hydrogen peroxide concentration: 0.1 μmol / ml; busulfan concentration: 0.01 μmol / ml. Spermatogenic layers of testicular organoarrays were cultured using media supplemented with either hydrogen peroxide or busulfan, and the proliferation level of spermatogonial stem cells was detected.

[0060] The results are as follows Figure 5 As shown in Figure E, compared with the control group, the proliferation of spermatogonial stem cells was reduced in the hydrogen peroxide group, indicating interference from oxidative stress. The reduction in spermatogonial stem cell proliferation was even more pronounced in the busulfan group. Therefore, the testicular organ-on-a-chip constructed in this invention exhibits significant sensitivity to drug activity or toxicity and can be used for pharmacological and toxicological screening.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A microfluidic-based testicular organ-on-a-chip, characterized in that, The testicular organ-on-a-chip includes microchannel unit A and microchannel unit B, a membrane structure, spermatogonial stem cells, testicular Sertoli cells, and testicular interstitial cells. The microchannel unit A and microchannel unit B each independently include an inlet microchannel, a culture microchannel, and an outlet microchannel; The microchannel unit A and microchannel unit B are located on both sides of the membrane structure, forming a sandwich structure; wherein the bottom of the two culture microchannels in microchannel units A and B are connected to both sides of the membrane structure. The membrane structure is coated with fibronectin and collagen IV on both sides; the membrane structure is a carbonate polyester filter membrane with a pore size of 0.4 μm; The spermatogonial stem cells and testicular Sertoli cells are located in the culture microchannel of microchannel unit A, forming a spermatogenesis layer; the spermatogonial stem cells adhere to the surface of the testicular Sertoli cells, and the testicular Sertoli cells adhere to the membrane structure at the bottom of the culture microchannel; the spermatogonial stem cells do not adhere to the membrane structure. The testicular interstitial cells are located in the culture microchannel of microchannel unit B, forming a nutrient layer; the testicular interstitial cells adhere to the membrane structure at the bottom of the culture microchannel; The nutrient layer delivers nutrients to the spermatogenic layer through a membrane structure; The seeding sequence of spermatogonial stem cells, testicular Sertoli cells, and testicular interstitial cells in the testicular organ-on-a-chip is as follows: First, testicular Sertoli cells are seeded in microchannel unit A and allowed to remain still for 3 days to allow the testicular Sertoli cells to adhere to the membrane structure; then, testicular interstitial cells are seeded in microchannel unit B and allowed to remain still for 3 days to allow the testicular interstitial cells to adhere to the membrane structure; finally, spermatogonial stem cells are seeded in microchannel unit A to allow the spermatogonial stem cells to adhere to the testicular Sertoli cells.

2. The testicular organ chip as described in claim 1, characterized in that, The widths of the inlet microchannel and the outlet microchannel are both 1 mm; The culture microchannel is 12 mm long, 2 mm wide, and 0.9 mm deep; The membrane structure is 20 mm long, 7 mm wide, and 0.5 mm thick.

3. The testicular organ chip as described in claim 1, characterized in that, It also includes an inlet microchamber and an outlet microchamber, the diameter of which is 1 mm; One end of the inlet microchamber is connected to the inlet microchannel, and the other end is connected to the culture medium container; One end of the outlet microchamber is connected to the outlet microchannel, and the other end is connected to the culture medium container.

4. The testicular organ chip as described in claim 1, characterized in that, The detection function module is formed by detecting the difference in composition between the input and output cell culture medium in the chip channel.

5. A method for preparing a testicular organ-on-a-chip as described in any one of claims 1-4, characterized in that, Includes the following steps: Step 1: Polydimethylsiloxane is heated and cured in a mold to obtain an integral structure in which two microchannel units are connected together; The microchannel culture site was cut into two parts, upper and lower, to obtain independent microchannel unit A and microchannel unit B. The inlet and outlet microchannels of the microchannel unit are punched and perforated to obtain the inlet and outlet microchannels of the microchannel unit. Step 2: Prepare the carbonated polyester filter membrane; Step 3: The culture microchannels of the two microchannel units of the testicular organ-on-a-chip from Step 1 are sandwiched with a polyester carbonate filter membrane to form a sandwich structure and then hot-pressed together to obtain the testicular organ-on-a-chip structure. Step 4: Spermatogonial stem cells, testicular Sertoli cells, and testicular interstitial cells are introduced into the culture microchannel through the inlet microchannel and then cultured. The specific steps for cell infusion in step 4 are as follows: (1) Testicular supporting cells were implanted into the culture microchannel of microchannel unit A and allowed to stand still, so that they adhered to one side of the membrane structure. (2) The testicular interstitial cells were implanted into the culture microchannel of microchannel unit B and allowed to stand still, so that they adhered to the other side of the membrane structure. (3) The spermatogonial stem cells are implanted into the culture microchannel of the microchannel unit A, and the spermatogonial stem cells adhere to the testicular supporting cells.

6. The method for preparing a testicular organ-on-a-chip as described in claim 5, characterized in that, It also includes adding a mixed coating of fibronectin and collagen IV to both sides of the membrane structure.

7. The application of the microfluidic-based testicular organ-on-a-chip as described in any one of claims 1-4 in drug screening.

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