A dual-stage chamber structure organoid culture microarray and its application method
By designing a two-level chamber structure for organoid culture microarrays, and combining it with PDMS and matrix gel, the problems of long-term stability and morphogenesis in organoid culture were solved, achieving uniform regulation and efficient culture of organoids, and improving experimental efficiency.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HARBIN INST OF TECH
- Filing Date
- 2026-04-20
- Publication Date
- 2026-07-14
AI Technical Summary
Existing microarray culture methods are difficult to achieve long-term stable culture of organoids and cannot reproduce the natural morphogenesis process in vivo, which leads to organoids being prone to decreased activity, structural abnormalities, or even apoptosis, and the experimental reproducibility is poor.
A two-chamber structure organoid culture microarray is designed, comprising a micropore array on the surface of a cylinder and a gel application tank. Each micropore in the micropore array is a stepped through-hole. Combined with the use of PDMS and matrix gel, a stable culture environment is formed.
It achieves uniform control of the initial culture state of organoids, ensures consistency throughout the development process, supports the stable introduction and retention of matrix gel, promotes the normal morphogenesis of organoids, solves the problems of consistency and insufficient nutrient supply in traditional culture methods, and improves experimental operation efficiency and potential for large-scale application.
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Figure CN122381920A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of organoid culture technology, specifically to an organoid culture microarray with a two-level chamber structure and its application method. Background Technology
[0002] Organoids are three-dimensional cellular structures that self-organize and develop from stem cells. Their core advantage lies in their ability to highly mimic physiological conditions in vivo, including cell polarity, intercellular connections, tissue-specific microstructures, and the reconstruction of structural features of primitive organs. Therefore, they have sparked widespread research revolutions in multiple fields such as tissue engineering, drug screening, and disease modeling.
[0003] In conventional laboratory settings, the most common method for creating organoids is to embed induced pluripotent stem cells or adult stem cells into extracellular matrices such as Matrigel gels for droplet culture. However, this traditional method suffers from several technical bottlenecks:
[0004] First, the organoids are randomly distributed in the three-dimensional space of the droplets, resulting in extremely poor consistency in their size and development process. This not only causes great inconvenience for subsequent dynamic observation and quantitative analysis, but also seriously affects the reliability of experimental data.
[0005] Secondly, the physical barrier properties of hydrogels make it difficult for nutrients to penetrate evenly, and the central region of organoids often dies due to insufficient nutrient supply, significantly reducing the success rate of culture.
[0006] Third, in applications such as drug screening and toxin detection, test compounds need to penetrate hydrogels to act on organoids. Uncontrollable variables such as diffusion delay and concentration decay exist in the process, which directly leads to poor experimental repeatability.
[0007] To address the challenge of organoid culture uniformity, current research has developed high-throughput culture technologies based on microarrays. This technology, by precisely controlling the initial cell count within each microwell, ensures a certain degree of organoid homogeneity. However, this type of culture operates in a purely suspended environment, lacking the structural support provided by an extracellular matrix (such as hydrogels) and the necessary microenvironment for the slow release of nutrients. This makes it difficult to achieve long-term stable culture of organoids and to replicate the natural morphogenesis process in vivo, significantly limiting their normal development and functional maturation. With prolonged culture periods, organoids are prone to decreased activity, structural abnormalities, and even apoptosis, severely restricting their application in biomedical research. Summary of the Invention
[0008] To address the challenges of existing microarray culture methods in achieving long-term stable culture of organoids and replicating the natural morphogenesis process in vivo, which can lead to decreased activity, structural abnormalities, and even apoptosis in organoids, this invention proposes a two-level chamber structure organoid culture microarray and its application method.
[0009] The technical solution adopted in this invention is:
[0010] A two-level chamber structure organoid culture microarray is provided. It is a cylinder with a micropore array at the center of the upper surface of the cylinder. The center point of the micropore array coincides with the central axis of the cylinder. The height of the micropore array is less than the height of the cylinder. The bottom surface of the micropore array to the bottom surface of the cylinder is a cavity. Multiple glue-adding grooves are machined in a ring on the outer side of the micropore array. The glue-adding grooves are connected to the cavity. Each micropore in the micropore array is a stepped through-hole with the diameter of the stepped through-hole increasing from top to bottom.
[0011] A method for applying a two-level chamber structured organoid culture microarray includes the following steps:
[0012] S1. Coat the lower surface of one or more organoid culture microarrays with PDMS, attach each PDMS-coated organoid culture microarray to each well of a 12-well plate, and then place the 12-well plate in a vacuum drying oven for curing.
[0013] S2. Place the cured 12-well plate into a plasma cleaner, introduce air, and perform plasma treatment for 150 seconds at room temperature.
[0014] S3. Sterilize the plasma-treated 12-well plate with ultraviolet light for 1 hour.
[0015] S4. Add 6 mg / ml of matrix gel to the gel filling tank of each organoid culture microarray in the sterilized 12-well plate until the matrix gel fills the cavity and the second well of each microwell. Place the 12-well plate with the added matrix gel in a cell culture incubator at 37°C for 40 min to obtain a cured 12-well plate.
[0016] S5. Based on the cured 12-well plate, add 1% bovine serum albumin to the first well of each microplate to fully soak the first well;
[0017] S6. Remove bovine serum albumin from each first well and rinse thoroughly with 1xPBS;
[0018] S7. Add mouse airway organoid suspension to the first well of each microwell and let stand for 10 min.
[0019] S8. Remove excess organoid suspension and rinse with 1xPBS until no organoid suspension remains on the surface of the organoid culture microarray.
[0020] S9. Based on S8, add culture medium mixed with 2% matrix gel to each well of the 12-well plate until the organoid culture microarray is completely infiltrated, and then place it in a cell culture incubator for culture to complete the application of the organoid culture microarray.
[0021] The beneficial effects of this invention are as follows:
[0022] 1. The microarray structure design for organoid culture enables uniform regulation of the initial culture state of organoids, thereby ensuring the consistency of their development throughout the entire process. PCR detection of key gene expression in organoids cultured for 14 days showed that, compared with conventional droplet culture, the variance in the expression levels of key genes in the organoids obtained by this invention was significantly smaller, effectively solving the core problem of inconsistent organoid development in traditional droplet culture.
[0023] 2. The stepped structure design of the micropores supports the stable introduction and retention of matrix gel, allowing organoids to be in full contact with matrix gel throughout the culture process. This not only uses the structural support provided by matrix gel to simulate the physiological microenvironment in vivo and promote the normal morphology of organoids, but also avoids the problem of insufficient nutrient supply in suspension culture through the nutrient slow-release properties of matrix gel, thus achieving long-term stable culture of organoids and breaking through the technical bottleneck of traditional microarray suspension culture, which is unable to support the long-term development of organoids.
[0024] 3. The organoid culture microarray of this invention has strong integration compatibility and can be directly adapted to conventional laboratory culture carriers such as 24-well plates and 12-well plates. High-throughput culture can be achieved without additional modification, which significantly improves experimental operation efficiency and large-scale application potential.
[0025] 4. This invention has broad cell adaptability and provides a universal culture program for different types of organoids, without the need to adjust the core structure design for specific organoids. Attached Figure Description
[0026] Figure 1 This is a schematic diagram of an organoid culture microarray;
[0027] Figure 2 This is a front view of the organoid culture microarray;
[0028] Figure 3 yes Figure 2 Cross-sectional view of AA in the middle;
[0029] Figure 4 yes Figure 2 Cross-sectional view of BB in the middle;
[0030] Figure 5 This is a schematic diagram of an organoid culture microarray integrated into a 12-well plate;
[0031] Figure 6This is a schematic diagram of a mold for organoid culture microarrays;
[0032] Figure 7 yes Figure 7 Cross-sectional view of CC in China;
[0033] Figure 8 This is a schematic diagram of organoid culture in an organoid culture microarray;
[0034] Figure 9 This is the initial state of organoids in organoid culture microarrays;
[0035] Figure 10 This is the state of organoids after 14 days of culture in an organoid culture microarray;
[0036] Figure 11 This is a comparison of gene expression differences between droplets and organoid microarrays cultured after 14 days of culture.
[0037] Figure 12 This is the state of organoids after 28 days of culture in an organoid culture microarray; Detailed Implementation
[0038] Specific implementation method one: Combining Figures 1-5 This embodiment describes a two-level chamber structure organoid culture microarray. For example... Figure 1 As shown, the organoid culture microarray is a cylinder 1. A micropore array 2 is positioned at the center of the upper surface of the cylinder 1, with the center point of the micropore array 2 coinciding with the central axis of the cylinder 1. The height of the micropore array 2 is less than the height of the cylinder 1. A cavity 11 is formed between the bottom surface of the micropore array 2 and the bottom surface of the cylinder 1. Multiple adhesive application grooves 3 are annularly machined on the outer side of the micropore array 2, and the adhesive application grooves 3 communicate with the cavity 11. Each micropore 21 in the micropore array 2 is a stepped through-hole, with the diameter of the stepped through-hole increasing sequentially from top to bottom.
[0039] In this embodiment, such as Figures 2-5As shown, cylinder 1 has a diameter of 19.5 mm and a height of 3.2 mm. The structure of cylinder 1 can be integrated into a general 12-well plate. The micropore array 2 is hexagonally distributed, and its height is approximately half the height of cylinder 1. The height of cavity 11 is 1.4 mm. Micropore array 2 is used to load cell-like structures, and cavity 11 is used to load hydrogel (matrix). A gel filling groove 3 is machined along the height direction of cylinder 1 on the upper, lower, left, and right sides of micropore array 2. The gel filling groove 3 has a rectangular structure, with a length and width of 2 mm and 1.85 mm, respectively, and its height extends from the upper surface to the bottom surface of cylinder 1. The gel filling groove 3 communicates with cavity 11 and is used to add hydrogel to fill the bottom of micropore array 2. The four gel filling grooves 3 can balance the air pressure, allowing the hydrogel to flow fully into micropore array 2. Each micropore 21 consists of a first pore 211 and a second pore 212 from top to bottom. The central axes of the first pore 211 and the second pore 212 coincide. The diameter of the first pore 211 is 0.6 mm and the height is 0.8 mm. The diameter of the second pore 212 is 0.8 mm and the height is 1 mm. The bottom of the second pore 212 is rounded outward instead of a right angle to facilitate the flow of the matrix adhesive. The center-to-center distance between adjacent micropores 21 in the micropore array 2 is 1.3 mm.
[0040] Specific implementation method two: such as Figure 6 and Figure 7 As shown, this embodiment differs from specific embodiment one in that the method for preparing a two-level chamber structure organoid culture microarray includes the following steps:
[0041] Step 1: Mix the PDMS prepolymer and crosslinking agent at a mass ratio or volume ratio of 10:1. After stirring evenly, place the mixture in a vacuum drying oven to degas until all air bubbles generated during the mixing process are completely eliminated.
[0042] Step 2: Fabricate the corresponding mold based on the organoid culture microarray structure proposed in Specific Implementation Method 1.
[0043] Step 3: Pour the PDMS, after removing air bubbles, into the mold and let it stand for a fixed time. Place the mold after pouring the PDMS into a vacuum drying oven to allow the PDMS to undergo a cross-linking reaction and cure. The curing temperature is 80℃ and the curing time is 4 hours.
[0044] Step 4: Cool the cured PDMS and gently peel it off from the mold to obtain the organoid culture microarray proposed in this invention.
[0045] The other steps and parameters are the same as in Specific Implementation Method 1.
[0046] Specific implementation method three: such as Figures 8 to 12As shown, this embodiment differs from specific embodiments one or two in that the application method of the organoid culture microarray with a two-level chamber structure includes the following steps:
[0047] S1. Coat the lower surface of one or more organoid culture microarrays with 50 μL of... PDMS is applied to each organoid culture microarray coated with PDMS and then attached to each well of a 12-well plate. The 12-well plate is then placed in a vacuum drying oven for curing. After curing, the organoid culture microarray will be firmly fixed in the 12-well plate.
[0048] S2. Place the cured 12-well plate into a plasma cleaner, introduce air, and perform plasma treatment for 150 seconds at room temperature.
[0049] S3. Disinfect the plasma-treated 12-well plate with ultraviolet light for 1 hour.
[0050] S4. Add 6 mg / ml of matrix gel to the gel application tank 3 of each organoid culture microarray in the sterilized 12-well plate until the matrix gel fills the cavity 11 and then flows into and fills the second well 212 of each microarray 21. Since the diameter of the first well 211 is smaller than that of the second well 212, and the junction of the upper and lower layers is a right angle structure, it will block the flow of matrix gel, allowing the matrix gel to fill only the second well 212 without entering the first well 211. Place the 12-well plate with the added matrix gel in a cell culture incubator at 37°C for 40 minutes to allow the matrix gel to fully cure, resulting in a cured 12-well plate.
[0051] S5. Based on the cured 12-well plate, 200 μL of precipitate is added to each first well 211 of each microwell 21. The first well 211 was thoroughly soaked in 1% bovine serum albumin at room temperature for 30 minutes to prevent cell adhesion.
[0052] S6. Remove bovine serum albumin from each well 211 and gently rinse well 211 with 1xPBS.
[0053] S7. Add mouse airway organoid suspension to each well 211 and let stand for 10 minutes. The organoids will deposit in the well 211 due to gravity and make full contact with the matrix gel in the well 212 below. In subsequent culture, each well will form an independent, highly consistent organoid. The organoids are in constant contact with the matrix gel during the culture process, providing structural support and necessary growth factors, which ensures the stability and consistency of the organoid culture over a long period of time.
[0054] S8. Remove excess organoid suspension and gently rinse with 1xPBS until no organoid suspension remains on the surface of the organoid culture microarray.
[0055] S9. Based on S8, add culture medium mixed with 2% matrix gel to each well of the 12-well plate to ensure complete immersion of the organoid culture microarray, and then place it in a cell culture incubator for culture to complete the application of the organoid culture microarray.
[0056] Other steps and parameters are the same as in specific implementation method one or two.
[0057] This invention may have other embodiments. Without departing from the spirit and essence of this invention, those skilled in the art can make various corresponding changes and modifications according to this invention, but these corresponding changes and modifications should all fall within the protection scope of the appended claims.
Claims
1. A two-level chamber structure organoid culture microarray, characterized in that: It is a cylinder (1), and a micro-hole array (2) is set at the center of the upper surface of the cylinder (1). The center point of the micro-hole array (2) coincides with the central axis of the cylinder (1). The height of the micro-hole array (2) is less than the height of the cylinder (1). The bottom surface of the micro-hole array (2) to the bottom surface of the cylinder (1) is a cavity (11). Multiple glue-adding grooves (3) are machined in a ring on the outer side of the micro-hole array (2). The glue-adding grooves (3) are connected to the cavity (11). Each micro-hole (21) in the micro-hole array (2) is a stepped through hole. The diameter of the stepped through hole increases from top to bottom.
2. The organoid culture microarray with a two-level chamber structure according to claim 1, characterized in that: The micropore array (2) is distributed in a regular hexagonal shape, and the height of the micropore array (2) is greater than or equal to half the height of the cylinder (1).
3. The organoid culture microarray with a two-level chamber structure according to claim 2, characterized in that: The outer side of the microporous array (2) is machined with four adhesive grooves (3), which are located on the upper side, lower side, left side and right side of the microporous array (2).
4. The organoid culture microarray with a two-level chamber structure according to claim 3, characterized in that: Each glue-adding groove (3) has a rectangular structure.
5. The organoid culture microarray with a two-level chamber structure according to claim 4, characterized in that: The bottom of each micropore (21) is rounded outwards.
6. The organoid culture microarray with a two-level chamber structure according to claim 5, characterized in that: Each micropore (21) is composed of a first pore (211) and a second pore (212) from top to bottom, and the central axes of the first pore (211) and the second pore (212) coincide.
7. The organoid culture microarray with a two-level chamber structure according to claim 6, characterized in that: Its preparation method includes the following steps: Step 1: Mix the PDMS prepolymer and crosslinking agent according to the mass ratio or volume ratio, stir evenly, and then put them into a vacuum drying oven to degas until all the air bubbles generated during the mixing process are completely eliminated. Step 2: Fabricate a mold for organoid culture microarrays; Step 3: Pour the PDMS that has been degassed in Step 1 onto the mold and let it stand for a fixed time. Then, place the mold with the poured PDMS into a vacuum drying oven for curing. Step 4: Cool the solidified PDMS to obtain organoid culture microarrays.
8. The organoid culture microarray with a two-level chamber structure according to claim 7, characterized in that: In step one, the mass ratio or volume ratio of PDMS prepolymer to crosslinking agent is 10:
1.
9. The organoid culture microarray with a two-level chamber structure according to claim 8, characterized in that: In step three, the curing temperature is 80℃ and the curing time is 4 hours.
10. A method for applying a two-level chamber structure organoid culture microarray, characterized in that: It includes the following steps: S1. Coat the lower surface of one or more organoid culture microarrays with PDMS, attach each PDMS-coated organoid culture microarray to each well of a 12-well plate, and then place the 12-well plate in a vacuum drying oven for curing. S2. Place the cured 12-well plate into a plasma cleaner, introduce air, and perform plasma treatment for 150 seconds at room temperature. S3. Sterilize the plasma-treated 12-well plate with ultraviolet light for 1 hour. S4. Add 6 mg / ml of matrix gel to the gel filling tank (3) of each organoid culture microarray on the sterilized 12-well plate until the matrix gel fills the cavity (11) and the second well (212) of each microwell (21). Place the 12-well plate with the matrix gel in a cell culture incubator at 37°C for 40 min to obtain the cured 12-well plate. S5. Based on the cured 12-well plate, add 1% bovine serum albumin to the first well (211) of each microwell (21) to fully soak the first well (211); S6. Remove bovine serum albumin from each first well (211) and rinse thoroughly with 1xPBS; S7. Add mouse airway organoid suspension to the first well (211) of each microwell (21) and let stand for 10 min; S8. Remove excess organoid suspension and rinse with 1xPBS until no organoid suspension remains on the surface of the organoid culture microarray. S9. Based on S8, add culture medium mixed with 2% matrix gel to each well of the 12-well plate until the organoid culture microarray is completely infiltrated, and then place it in a cell culture incubator for culture to complete the application of the organoid culture microarray.