An organoid chip that simulates the immune microenvironment of multiple affected organs in endometriosis

Abstract

This invention relates to an organoid microarray that simulates the immune microenvironment of multiple organs affected by endometriosis. The microarray comprises a chip with three spatially independent cell culture chambers connected by a series channel: an ovarian organoid culture chamber, a peritoneal culture chamber, and an intestinal organoid culture chamber. Each cell culture chamber has a fluid channel in both its upper and lower layers. This invention also provides a method for organ culture using the organoid microarray that simulates the immune microenvironment of multiple organs affected by endometriosis. Its advantages include: simultaneously connecting ectopic endometrial organoids with three target organs; simultaneously simulating the implantation and invasion process of shed endometrial tissue under the influence of the immune microenvironment and in different organs; and constructing a mixed organoid including glandular and stromal cells to explore the interactions and influences between cells of multiple organs and tissues. This is of great significance for preventing endometriosis from affecting vital organs and for discovering new therapeutic targets.

Description

[Technical Field]

[0001] This invention relates to the field of organ-on-a-chip technology, specifically to an organ-on-a-chip that simulates the immune microenvironment of multiple affected organs in endometriosis. [Background Technology]

[0002] Endometriosis (EMs), also known as endometriosis, has an incidence rate of 10-15% in women of reproductive age, affecting approximately 180 million women worldwide. Its lesions are widespread and diverse, most commonly affecting the ovaries, but can also involve the peritoneum and even extend beyond the peritoneum to form lesions in the rectovaginal septum, causing a range of clinical symptoms such as pain, infertility, and pelvic masses. It can also invade vital organs such as the intestines, affecting their function. Currently, treatments using medication, surgery, or a combination of both have a high recurrence rate, seriously endangering women's health. However, the pathogenesis of EMs remains unclear, and while some patients only have ovarian involvement, others experience involvement of multiple other organs. Many mysteries remain, and in-depth research into these issues is crucial for preventing EMs from affecting vital organs and for discovering new therapeutic targets.

[0003] According to the classic retrograde menstruation theory of endometrial stenosis (EMs), the first step in lesion formation is the adhesion of the endometrium to the peritoneum of the pelvic cavity or the surface of organs such as the ovaries and intestines. However, retrograde menstruation exists in 90% of women of childbearing age and is almost a physiological phenomenon, yet only 10-15% of them develop EMs. Therefore, this process requires overcoming the body's own peritoneal microenvironment defenses. It has been established that immune tolerance and immune escape exist in the peritoneal microenvironment of EMs patients, and macrophages are one of the most critical cellular components of the body's peritoneal immune system. In recent years, the role of immune factors in the pathogenesis of EMs has received attention. At the same time, whether there are differences in the causes of EMs lesions in different locations and whether there is a correlation between the pathogenesis of multiple affected organs remain unsolved mysteries. There is an urgent need to develop research models that can simulate the immune microenvironment of EMs and more closely approximate the actual situation in the body of EMs patients.

[0004] To date, in vitro cell models of EMs have only targeted ectopic endometrial stromal cells and lack glandular epithelial cell components. Although transwell can achieve co-culture of two types of cells, it cannot simulate the interaction and mutual influence between cells of multiple organs and tissues, nor has it achieved the simulation of the immune microenvironment. Multi-organ-on-a-chip technology is the key to solving these problems.

[0005] Chinese patent application CN112280678A discloses a detachable and reusable hydrophobic or superhydrophobic microfluidic organ-on-a-chip. It utilizes hydrophobic or superhydrophobic surfaces with low critical surface tension to construct microfluidic organ-on-a-chips, specifically for constructing heart, liver, brain, tumor, kidney, intestinal, skin, fat, blood vessel, uterus, eye, nose, bone, periodontal, pancreatic, spleen, placenta, lung, muscle, larynx, bone marrow, diabetes, and multi-organ chips. The organ-on-a-chip constructed using this invention is detachable and reusable, significantly reducing the application cost of microfluidic organ-on-a-chips. However, this patent's organ-on-a-chip primarily simulates the structure and function of the uterus itself, but cannot simulate the invasive function of the endometrium in the immune microenvironment outside the uterus, i.e., the influence of various cytokines on the invasion of the endometrium in multiple organ tissues. Consequently, it cannot simulate a research model that more closely resembles the actual situation in patients with endometriosis (EMS).

[0006] In summary, there have been reports on multi-organ-on-a-chip technology, but there are no relevant literature or patents on pathological models of endocrine disorders (EMs), especially organ-on-a-chip technology that simulates the immune microenvironment of multiple affected organs in EMs. [Summary of the Invention]

[0007] The purpose of this invention is to provide a pathological model for EMs, especially an organoid chip that simulates the immune microenvironment of multiple affected organs in EMs.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] An organoid chip simulating the immune microenvironment of multiple affected organs in endometriosis includes a chip containing three spatially independent cell culture chambers connected by a series channel: an ovarian organoid culture chamber, a peritoneal culture chamber, and an intestinal organoid culture chamber. The bottom of the ovarian organoid culture chamber is composed of micropits. The peritoneal culture chamber is used to construct peritoneal biomimetic tissue. The bottom of the intestinal organoid culture chamber is composed of micropillars. The upper and lower layers of the series channel are composed of porous membranes. Each cell culture chamber has a fluid channel in both the upper and lower layers, which is connected to the series channel through the porous membrane. Patient-derived peritoneal fluid is added to simulate the immune microenvironment.

[0010] As a preferred example, the chip material can be prepared from biocompatible materials such as PMMA, PDMS, and PC.

[0011] As another preferred embodiment, the cell culture chamber is composed of hexagons, with a width of 8-10 mm and a total area of ​​1-2 cm². 2 There are entrances and exits at both ends.

[0012] As another preferred embodiment, the bottom of the ovarian organoid culture chamber has 12 micropits with a diameter of 100-600 μm and a depth of 100-400 μm. The micropits are U-shaped, polygonal, etc., and are used to culture ovarian organoids. There are entrances and exits at both ends.

[0013] As another preferred embodiment, the bottom of the intestinal organ culture chamber consists of 12 micropillars with a diameter of 200-400 μm and a spacing of 50-150 μm. The height of the micropillars is consistent with the depth of the culture chamber and they are in contact with the upper membrane. There are inlets and outlets at both ends.

[0014] As another preferred embodiment, the serial channels form U-shaped structures in corresponding areas of the cell culture chamber, with a width of 1-1.5 mm and a spacing of 1-2 mm between the U-shaped areas. The U-shaped areas are respectively connected to the ovarian organoid culture chamber, the peritoneal culture chamber, and the intestinal organoid culture chamber through porous membranes.

[0015] As another preferred embodiment, a liquid storage pool with a height of 1-1.5cm is provided on both sides of the upper fluid channel. The top of the liquid storage pool is sealed with a cover of a sterile 0.22-micron filter element. The flow between the liquids is promoted by swaying to simulate the dynamic microenvironment of the peritoneal and pelvic fluids. The swaying angle is 15-60° and the swaying speed is 10-30 times / minute.

[0016] To achieve the second objective mentioned above, the technical solution adopted by the present invention is as follows:

[0017] A method for organ culture using an organoid chip that simulates the immune microenvironment of multiple affected organs in endometriosis, comprising any of the organoid chips described above that simulate the immune microenvironment of multiple affected organs in endometriosis, and the following steps.

[0018] a) First, sterilize and disinfect the chip for later use;

[0019] b) Modify the cell culture chamber and the series channel in the middle composed of a porous membrane with matrix gel; seed the ovarian surface epithelial cells derived from the patient into the membrane side of the ovarian organoid culture chamber, and after adhesion, seed follicular cells into the micropits, gently remove the culture medium, and then infuse the micropits with 3D matrix gel to ensure that the micropits are filled with matrix gel, but not the entire culture chamber, and add culture medium after solidification;

[0020] c) Peritoneal mesothelial cells are cultured on the membranous side of the peritoneal culture chamber to represent peritoneal tissue;

[0021] d) Intestinal organoid epithelial cells derived from human induced pluripotent stem cells were seeded on the membrane side of the intestinal organoid culture chamber to form intestinal epithelial structures on the membrane side and micropillar surface;

[0022] e) After all cell culture chambers have been seeded, flip the chip and seed the ectopic endometrial organoids and matrix gel mixture from EMs patients into the serial channel.

[0023] f) After the intermediate layer has solidified, culture medium and patient-derived peritoneal fluid are perfused into the uppermost fluid channel;

[0024] g) Seal all inlets and outlets with a cover containing a filter cartridge and use a shaker for dynamic cultivation;

[0025] h) Record the invasion of ectopic endometrial organoids into three organs at different times.

[0026] The advantages of this invention are:

[0027] This invention connects ectopic endometrial organoids with three target organs simultaneously, simulating the implantation and invasion of shed endometrial tissue into different organs under the influence of the immune microenvironment. It also constructs a mixed organoid including glands and stromal cells to realistically simulate shed endometrial tissue. Regular fluid stimulation of the ectopic endometrial organoids simulates the dynamic microenvironment of the abdominal and pelvic cavities, making the entire model more closely resemble the actual situation in ectopic endometrial patients. This allows for the exploration of interactions and influences between multiple organ tissues and cells, enabling analysis of differences in the causes of ectopic endometrial lesions in different locations and the correlation between the pathogenesis of multiple affected organs. This is of great significance for preventing ectopic endometrial lesions from affecting vital organs and for discovering new therapeutic targets. [Attached Image Description]

[0028] Appendix Figure 1 This is a schematic diagram of how shed endometrial tissue implants in the immune microenvironment and invades target organs.

[0029] Appendix Figure 2 This is a design diagram of an organoid chip that simulates the immune microenvironment of multiple affected organs in endometriosis according to the present invention.

Detailed Implementation Methods

[0030] The present invention will now be further described with reference to the embodiments and the accompanying drawings.

[0031] The reference numerals and components involved in the accompanying drawings are shown below:

[0032] 1. Chip 2. Cell culture chamber 21. Ovarian organoid culture chamber 211. Minor pit 22. Peritoneal Culture Chamber 23. Intestinal organoid culture chamber 231. Microcolumn 3. Series channel 4. Fluid Channel

[0033] Example 1

[0034] Please see Figure 1 and Figure 2 , Figure 1 This is a schematic diagram illustrating how shed endometrial tissue implants in the immune microenvironment and invades target organs. Figure 2 This is a design diagram of an organoid chip that simulates the immune microenvironment of multiple affected organs in endometriosis according to the present invention.

[0035] See Figure 1 , Figure 1 This paper succinctly explains the classic retrograde menstruation theory of endometrial stenosis (EMs). Most women experience retrograde menstruation, but only a minority develop EMs. Furthermore, the relationship between EMs and retrograde menstruation remains unclear. This application can realistically simulate the dynamic changes of shed endometrial tissue in the immune microenvironment. The entire model closely resembles the actual situation in EMs patients, which helps to explore the interactions and influences between cells in multiple organs and tissues. This allows for the analysis of whether there are differences in the causes of EMs lesions in different locations and whether there is a correlation between the pathogenesis of multiple affected organs.

[0036] See Figure 2 The organoid chip simulating the immune microenvironment of multiple affected organs in endometriosis includes a chip 1. The chip 1 contains three spatially independent cell culture chambers 2 connected by a series channel 3. Preferably, the cell culture chambers 2 are hexagonal, with a width of 8-10 mm and a total area of ​​1-2 cm². 2 The chambers are designated as follows: ovarian organoid culture chamber 21, peritoneal culture chamber 22, and intestinal organoid culture chamber 23. The bottom of the ovarian organoid culture chamber 21 is composed of micropits 211, preferably 12 micropits 211 at the bottom. The micropits 211 have a diameter of 100-600 μm, a depth of 100-400 μm, and are U-shaped, polygonal, or similar in shape, and are used for culturing ovarian organoids. The peritoneal culture chamber 22 is used to construct peritoneal biomimetic tissue. The intestinal organoid culture chamber 23... The bottom of chamber 23 is composed of micropillars 231. Preferably, the bottom of the intestinal organ culture chamber 23 is composed of 12 micropillars 231. The micropillars 231 have a diameter of 200-400 μm and a spacing of 50-150 μm. The height of the micropillars 231 is consistent with the depth of the culture chamber and is in contact with the upper membrane. The upper and lower parts of the series channel 3 are composed of porous membranes. The upper and lower parts of the cell culture chamber 2 are each provided with a fluid channel 4, which is connected to the series channel 3 through a porous membrane. Peritoneal fluid from patients is added to simulate the immune microenvironment.

[0037] In this embodiment, the chip 1 is preferably made of biocompatible materials such as PMMA, PDMS, and PC.

[0038] In this embodiment, preferably, the serial channels 3 form U-shaped structures in corresponding areas within the cell culture chamber 2, with a width of 1-1.5 mm and a spacing of 1-2 mm between the U-shaped areas. The U-shaped areas are respectively connected to the ovarian organoid culture chamber 21, the peritoneal culture chamber 22, and the intestinal organoid culture chamber 23 through porous membranes.

[0039] In this embodiment, preferably, a liquid storage pool with a height of 1-1.5cm is provided on both sides of the upper fluid channel (not shown in the figure). The top of the liquid storage pool is sealed with a cover of a sterile 0.22-micron filter element. The flow between the liquids is promoted by swaying to simulate the dynamic microenvironment of the peritoneal and pelvic fluids. The swaying angle is 15-60° and the swaying speed is 10-30 times / minute.

[0040] It should be noted that this invention connects ectopic endometrial organoids with three target organs simultaneously, which can simultaneously simulate the implantation and invasion process of shed endometrial tissue in different organs under the influence of the immune microenvironment. It also constructs a mixed organoid including glands and stromal cells to realistically simulate shed endometrial tissue. By regularly stimulating the ectopic endometrial organoids with fluid, it simulates the dynamic microenvironment of the abdominal and pelvic cavities, making the entire model closer to the real situation in ectopic endometrial patients. It explores the interaction and mutual influence between multiple organ tissue cells, thereby allowing for the association between the different causes of ectopic endometrial lesion formation and the pathogenesis of multiple affected organs. This is of great significance for preventing ectopic endometrial tissue from affecting important organs and for discovering new therapeutic targets.

[0041] Example 2

[0042] To achieve the second objective mentioned above, the technical solution adopted by the present invention is as follows:

[0043] A method for organ culture using an organoid chip that simulates the immune microenvironment of multiple affected organs in endometriosis, comprising any of the organoid chips described above that simulate the immune microenvironment of multiple affected organs in endometriosis, and the following steps.

[0044] a) First, sterilize and disinfect chip 1 for later use;

[0045] b) Modify the cell culture chamber 2 and the series channel 3 in the middle, which is composed of a porous membrane, with matrix gel; seed the ovarian surface epithelial cells derived from the patient into the membrane side of the ovarian organoid culture chamber 21, and after adhesion, seed follicular cells into the micropits 211, gently remove the culture medium, and then infuse the micropits 211 with 3D matrix gel, ensuring that the micropits 211 are filled with matrix gel, but not the entire culture chamber, and add culture medium after solidification;

[0046] c) Peritoneal mesothelial cells representing peritoneal tissue were cultured on the membranous side of peritoneal culture chamber 22;

[0047] d) Intestinal organoid epithelial cells derived from human induced pluripotent stem cells were seeded on the membrane side of the intestinal organoid culture chamber 23 to form intestinal epithelial structures on the membrane side and the surface of micropillar 231;

[0048] e) After all cell culture chambers 2 have been inoculated, flip chip 1 and inoculate the mixture of ectopic endometrial organoids and matrix gel derived from EMs patients into the serial channel 3.

[0049] f) After the intermediate layer has solidified, culture medium and patient-derived peritoneal fluid are perfused into the uppermost fluid channel 4;

[0050] g) Seal all inlets and outlets with a cover containing a filter cartridge and use a shaker for dynamic cultivation;

[0051] h) Record the invasion of ectopic endometrial organoids into three organs at different times.

[0052] In summary, this invention utilizes organoid chip technology derived from patient-derived tissues and human stem cells to simulate the pathological process of EMs forming lesions in different organs under the influence of the immune microenvironment. This provides a new model for the study of EMs pathogenesis and drug development, and offers a better chip platform for the research of basic scientific questions and the screening of therapeutic drugs, thereby making the implementation of personalized medicine more feasible.

[0053] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. An organ-on-a-chip that simulates the immune microenvironment of multiple affected organs in endometriosis, characterized in that, The device includes a chip containing three spatially independent cell culture chambers connected by a series channel: an ovarian organoid culture chamber, a peritoneal culture chamber, and an intestinal organoid culture chamber. The bottom of the ovarian organoid culture chamber is composed of micropits. The peritoneal culture chamber is used to construct peritoneal biomimetic tissue. The bottom of the intestinal organoid culture chamber is composed of micropillars. The series channel is composed of porous membranes on both the upper and lower layers. Each cell culture chamber has a fluid channel on both the upper and lower layers, which is connected to the series channel through the porous membrane. Patient-derived peritoneal fluid is added to simulate the immune microenvironment.

2. The organ-on-a-chip that simulates the immune microenvironment of multiple affected organs in endometriosis according to claim 1, characterized in that, The chip material can be prepared from biocompatible materials such as PMMA, PDMS, and PC.

3. The organ-on-a-chip that simulates the immune microenvironment of multiple affected organs in endometriosis according to claim 1, characterized in that, The cell culture chamber is composed of hexagons, with a width of 8-10 mm and a total area of ​​1-2 cm². 2 There are entrances and exits at both ends.

4. The organ-on-a-chip that simulates the immune microenvironment of multiple affected organs in endometriosis according to claim 1, characterized in that, The bottom of the ovarian organoid culture chamber has 12 micropits, each with a diameter of 100-600 μm and a depth of 100-400 μm. The micropits are U-shaped, polygonal, or similar in shape and are used to culture ovarian organoids. There are entrances and exits at both ends.

5. The organoid chip according to claim 1, which simulates the immune microenvironment of multiple affected organs in endometriosis, is characterized in that... The bottom of the intestinal organ culture chamber consists of 12 micropillars with a diameter of 200-400 μm and a spacing of 50-150 μm. The height of the micropillars is consistent with the depth of the culture chamber and they are in contact with the upper membrane. There are inlets and outlets at both ends.

6. The organ-on-a-chip that simulates the immune microenvironment of multiple affected organs in endometriosis according to claim 1, characterized in that, The series channels form U-shaped structures in corresponding areas within the cell culture chambers, with a width of 1-1.5 mm and a spacing of 1-2 mm between the U-shaped areas. The U-shaped areas are connected to the ovarian organoid culture chamber, peritoneal culture chamber, and intestinal organoid culture chamber through porous membranes.

7. The organ-on-a-chip that simulates the immune microenvironment of multiple affected organs in endometriosis according to claim 1, characterized in that, The upper fluid channel has a liquid storage pool with a height of 1-1.5cm on both sides. The top of the liquid storage pool is sealed with a cover of a sterile 0.22-micron filter element. The flow between the liquids is promoted by swaying to simulate the dynamic microenvironment of the abdominal and pelvic cavities. The swaying angle is 15-60° and the swaying speed is 10-30 times / minute.

8. A method for organ culture using organ-on-a-chip technology that simulates the immune microenvironment of multiple affected organs in endometriosis, characterized in that... The organoid chip, which simulates the immune microenvironment of multiple affected organs in endometriosis as described in any one of claims 1-7, and the following steps; a) First, sterilize and disinfect the chip for later use; b) Modify the cell culture chamber and the series channel in the middle composed of a porous membrane with matrix gel; seed the ovarian surface epithelial cells derived from the patient into the membrane side of the ovarian organoid culture chamber, and after adhesion, seed follicular cells into the micropits, gently remove the culture medium, and then infuse the micropits with 3D matrix gel to ensure that the micropits are filled with matrix gel, but not the entire culture chamber, and add culture medium after solidification; c) Peritoneal mesothelial cells are cultured on the membranous side of the peritoneal culture chamber to represent peritoneal tissue; d) Intestinal organoid epithelial cells derived from human induced pluripotent stem cells were seeded on the membrane side of the intestinal organoid culture chamber to form intestinal epithelial structures on the membrane side and micropillar surface; e) After all cell culture chambers have been seeded, flip the chip and seed the ectopic endometrial organoids and matrix gel mixture from EMs patients into the serial channel. f) After the intermediate layer has solidified, culture medium and patient-derived peritoneal fluid are perfused into the uppermost fluid channel; g) Seal all inlets and outlets with a cover containing a filter cartridge and use a shaker for dynamic cultivation; h) Record the invasion of ectopic endometrial organoids into three organs at different times.

Images