Method and device for culturing organoid based on capillary tube

Abstract

The invention provides a method and a device for culturing an organoid based on a capillary tube, and belongs to the technical field of organoid culture. The method comprises the following steps: uniformly mixing a to-be-cultured cell cluster precipitate and diluted matrigel to form a suspension, and injecting the suspension into a capillary tube by utilizing capillary action; then putting the capillary tube into a cell culture box, taking the capillary tube out of the cell culture box after the suspension in the capillary tube is naturally solidified, filling the upper part of the solidified suspension with an organoid complete medium, then hanging the capillary tube on a bracket and putting the capillary tube into the cell culture box for culturing, and continuously updating the organoid complete medium to obtain the organoid complete medium. The dynamic stable growth of the organoid in the capillary tube is realized. According to the method, stable and dynamic culture of the organoids can be achieved, the requirement for supporting the three-dimensional environment in the growth process of the organoids can be met, nutrient substances can be sufficiently taken in, and metabolite of the organoids can be removed.

Description

Technical Field

[0001] This invention relates to the field of organoid culture technology, and in particular to a method and apparatus for capillary-based organoid culture. Background Technology

[0002] In recent years, organoid culture technology has made significant progress. Its rapid development in disease construction, drug development, personalized treatment, and multidisciplinary collaboration has broadened the application prospects of organoid technology and opened up new avenues for treating human diseases and regenerative medicine.

[0003] Organoids are tissue analogs with a certain spatial structure formed by in vitro 3D culture of adult stem cells or pluripotent stem cells. They have the cell types and similar spatial structures of the corresponding tissues and organs, and can simulate real organs in structure and function. They can also be stably passaged, hence they are also called miniature organs.

[0004] In organoid culture, the conventional method is to use well plates. While traditional well plate culture allows organoids to grow continuously in a three-dimensional environment with the supply of nutrients, this static culture is not conducive to the balanced uptake and absorption of nutrients by the organoids. This leads to significant differences in the shape and size of the organoids, and the metabolic waste produced cannot be eliminated in a timely manner, thus affecting organoid growth. Well plate culture also consumes a considerable amount of reagents and consumables, and the static culture has a very negative impact on the growth environment and growth status of organoids.

[0005] Capillaries are commonly used for liquid transport and can also be used to separate cells, cell clusters, spheroids, organoids, and small colonies. Small-diameter capillaries can target and pick up single cells without cross-contamination from adjacent cells. Larger-diameter capillaries can pick up cell clusters or spheroids without generating destructive shear forces, thus maintaining the integrity of the picked cells. The primary applications of capillaries are the separation of rare single cells, such as circulating tumor cells or fetal cells, and the separation of specific portions of single-cell clones and stem cell colonies for cell line development. They can also be used as delivery systems or microreactors for various substances. Summary of the Invention

[0006] The purpose of this invention is to provide a method and apparatus for culturing organoids based on capillary tubes. This method utilizes capillary tubes to achieve dynamic and stable culture of organoids, which is conducive to the uniform diffusion and flow of the complete organoid culture medium, facilitating the healthy and stable growth of organoids. It provides reference value and preliminary foundation for subsequent drug screening and clinical medical research and development of organoids.

[0007] This invention is achieved through the following technical solution. A method for culturing organoids based on capillary tubes involves mixing the cell mass precipitate to be cultured with a diluted matrix gel containing complete organoid culture medium to form a suspension. The suspension is then injected into a capillary tube using capillary action. The capillary tube is then placed in a cell culture incubator. After the suspension in the capillary tube has naturally solidified, the capillary tube is removed from the incubator. Complete organoid culture medium is then filled onto the solidified suspension, and the tube is suspended on a support and placed in a cell culture incubator for cultivation. The complete organoid culture medium continuously infiltrates into the suspension and diffuses and flows evenly. As the culture time increases and the organoids continue to grow, they continuously consume the nutrients in the complete organoid culture medium. By timely updating and replenishing the complete organoid culture medium, dynamic and stable growth of the organoids in the capillary tube is achieved.

[0008] Furthermore, the organoid complete culture medium is updated by injecting new organoid complete culture medium into the capillary every 24 hours.

[0009] Furthermore, the capillary is a "┌"-shaped capillary with a horizontal segment and a vertical segment. The suspension is injected into the end of the vertical segment through capillary action. After natural solidification in the cell culture incubator, the organoid complete culture medium is filled into the cavity above the suspension and the horizontal segment through the connecting tubing in the syringe. Then it is suspended on the support and placed in the cell culture incubator for continued culture.

[0010] The present invention also provides an apparatus for implementing a capillary-based method for culturing organoids, comprising a scaffold and a capillary tube suspended on the scaffold and placed in a cell culture incubator.

[0011] Specifically, the capillary is made of one of the following materials: glass, quartz, or polymer.

[0012] Specifically, the capillary tube is circular or square in shape.

[0013] Specifically, the inner diameter of the capillary is 0.9-1.1 mm.

[0014] Specifically, the support structure consists of two identical base columns on the left and right and an upper plate. The two base columns support the upper plate, and the upper plate has several holes or clamping structures for fixing capillaries.

[0015] The beneficial effects of the above-described technical solution of the present invention are as follows: This invention proposes a dynamic organoid culture system, which, compared with static culture in well plates, facilitates the balanced uptake of nutrients and timely removal of metabolic waste by organoids in capillaries. Through the continuous diffusion and flow of the complete organoid culture medium, the uniformity of organoid size is ensured, and dynamic and stable growth of organoids is achieved.

[0016] The capillaries used in this invention are commercially available. Using capillaries of different forms as carriers for organoid culture is inexpensive, readily available, and space-saving. This saves on experimental consumables and simplifies the method. Glass capillaries possess high hardness, strong heat and pressure resistance, and good chemical stability; they do not swell and are resistant to chemical corrosion. They are also highly resistant to common organic reagents. Using them to culture organoids allows for easier observation of organoid morphology under a microscope, avoiding many problems.

[0017] Organoids can be cultured dynamically throughout the entire process and simulated under microenvironmental conditions in capillaries. This is beneficial for the balanced uptake of nutrients and timely removal of metabolic waste by organoids in capillaries. Through the continuous diffusion and flow of the complete organoid culture medium, dynamic, stable and sustainable growth of organoids can be achieved. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the device for capillary culture of organoids in Embodiment 1 of the present invention; Figure 2 This is a morphological diagram of a mature small intestinal organoid that grows from a small intestinal crypt in Example 2; Figure 3 This is a diagram showing the uniformity of small intestinal organoid growth in the capillaries in Example 2; Figure 4 Immunohistochemical identification image of small intestinal organoids in capillaries in Example 2; In the diagram: 1-scaffold, 2-capillary, 3-organoid complete culture medium, 4-suspension. Detailed Implementation

[0019] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and embodiments. Example 1

[0020] A device based on capillary culture of organoids, such as Figure 1As shown, the support 1 consists of two identical base pillars on the left and right sides and an upper plate. The upper plate can fix and suspend one or more capillaries with different inner diameters, thus suspending the capillaries 2 and facilitating the stable and dynamic culture of organoids within the capillaries 2. The capillaries 2 have a circular cross-section and an inner diameter of 1.0 mm. The capillaries 2 are "┌"-shaped capillaries with horizontal and vertical sections, the horizontal section being 16 cm long and the vertical section 4 cm long. The capillaries 2 are made of glass, but quartz or polymer materials can also be used, as long as they meet the requirements of biocompatibility, non-cytotoxicity, and suitability for the three-dimensional culture microenvironment. The inner diameter of the capillaries 2 can be between 0.9 and 1.1 mm, or a square cross-section can be used. In this embodiment, a curved capillary is used to obtain a constant flow rate. The flow rate of the solution in the capillary is determined by the height difference between the lower outlets of the horizontal and vertical sections, which was experimentally determined to be 4 cm in length for the vertical section. The part of the capillary that needs to be bent is heated until it softens to a certain extent, then bent and cooled to room temperature. Example 2

[0021] A method for culturing organoids using capillary tubes, comprising the following steps: Step 1: Preparation of the required cell pellet and diluted matrix gel from organoid complete culture medium to form a suspension: Six- to eight-week-old normal male C57BL / 6 mice were intraperitoneally anesthetized and euthanized by cervical dislocation. Under aseptic conditions, approximately 3-20 cm of small intestine tissue was harvested and placed in pre-cooled, sterile, calcium-free medium. 2+ Mg 2+ The contents were thoroughly washed in phosphate-buffered saline (DPBS), divided into several small segments, and villi were scraped off. After washing, the tissue was cut into fragments of about 3 mm and digested at 4°C for 20-30 min. Afterward, the tissue was washed, filtered, and centrifuged (300×). g (5 min), the supernatant was aspirated to obtain cell clumps; a diluted matrix gel was composed of 80% matrix gel and 20% organoid complete culture medium 3; then the cell clumps were mixed with the diluted matrix gel to prepare suspension 4. The matrix gel purchased was from bioGenous, a biogenotechnology company, whose core components are laminin, type IV collagen, nestin, and heparan sulfate proteoglycan. In this application, experiments were conducted using matrix gel of different concentrations, and the volume percentage of matrix gel was finally determined to be 80%. Moreover, through experiments with filling lengths of 1.5 cm, 2.0 cm, 2.5 cm, and 3.0 cm in the vertical section of suspension 4, the initial position of organoid complete culture medium 3 was determined based on the initial color of the initial position, thereby determining the flow rate of organoid complete culture medium 3, and finally selecting the scheme of filling length of 1.5 cm in the vertical section of suspension 4.

[0022] Step 2: Method of culturing intestinal organoids using capillary tube 2: A suitable amount of suspension 4 is injected into the vertical end of capillary tube 2 under low temperature conditions. After solidification in a cell culture incubator for 15-20 minutes, the tube is removed. Organoid complete culture medium 3 is then filled above the suspension 4 and suspended on the upper plate of support 1 (Example 1). The tube is then placed in a cell culture incubator for further culture. The organoid complete culture medium 3 continuously infiltrates into the matrix gel-embedded suspension 4, spreading and flowing evenly. The organoid complete culture medium 3 flows at approximately 10... -6 The organoids flow at a rate of m / s. The organoids continuously consume the nutrients in the organoid complete culture medium 3. The remaining products and metabolites in the organoid complete culture medium 3 flow out from the lower end of the vertical section. Every 24 hours, new organoid complete culture medium is injected into the capillary 2 to ensure that the organoid complete culture medium 3 is updated in a timely manner. This keeps the growth environment of the organoids constantly updated rather than in a closed and static state, which helps to achieve dynamic and stable culture of the desired organ. The complete organoid culture medium consisted of DMEM / F12 medium (Durbeco modified Eagle medium / Ham nutrient mixture F-12) supplemented with N-acetylcysteine, B+ supplement, GlutaMAX (stable glutamine additive), 60 U / mL penicillin, 0.1 g / L streptomycin, 5 µg / L EGF (epidermal growth factor), 250 µg / L R-spondin-1 (R-chondrin 1), 100 µg / L Noggin, 500 nmol / L A83-01, 5 µmol / L Y-27632 and 5 mmol / L nicotinamide.

[0023] The morphological growth of small intestinal organoids was observed daily using an inverted microscope, showing their development from closed, round, juvenile U-shaped intestinal crypts within capillaries to mature, sprouting intestinal organoids. The morphological changes in these organoids were as follows: Figure 2 As shown, Figure 2 In the middle, A represents the intestinal crypt (initial state). Figure 2 B represents juvenile intestinal organs (which are closed-loop and round). Figure 2 C represents a mature intestinal organoid (with budding structures). Figure 2 This demonstrates that capillary culture can simulate the natural growth process, and the organoid morphology gradually becomes more complex, conforming to the laws of in vivo development.

[0024] In traditional well plates, nutrients in the culture medium are passively absorbed by the intestinal organoids through osmotic diffusion. This results in better growth of intestinal organoids at the periphery, while those in the core area die faster due to insufficient nutrient access. The device in this embodiment, however, allows for continuous and uniform flow and diffusion, enabling intestinal organoids to fully absorb nutrients from the culture medium. This solves the problems encountered with static well plates, resulting in more uniform intestinal organoid growth and ensuring consistent size. Figure 3As shown, by comparing static orifice plates ( Figure 3 (A) and dynamic capillary ( Figure 3 The growth of midgut organoids in B) visually demonstrates the advantages of capillary culture: organoids in static plates are of varying sizes and unevenly distributed, while organoids in capillaries are of uniform size and neatly arranged, proving that dynamic culture can promote balanced nutrient intake and metabolic waste excretion.

[0025] Step 3: Identification of intestinal organoids by immunohistochemical staining: Transfer the passaged suspension 4 from the capillary tube to an EP tube, add an appropriate amount of DPBS, centrifuge by pipetting, discard the supernatant, add 4% PFA and fix at 4℃ for 1 hour, then perform embedding, dehydration, permeabilization, paraffin embedding, freeze-coagulation, sectioning, dewaxing, dehydration, antigen retrieval, blocking, add diluted primary antibody and incubate overnight at 4℃, recover the primary antibody and add diluted secondary antibody and incubate at 37℃ for 1-2 hours, wash, add DAB staining solution and hematoxylin staining, differentiate with 1% hydrochloric acid and ethanol, dehydrate and clear with graded ethanol, air dry and mount with mounting glue.

[0026] Microscopic observation revealed that the panthenocyte marker LYZ, unique to small intestinal organoids, was expressed as a brownish-yellow color, indicating high expression. This immunohistochemical staining further confirms that our device can successfully culture mature intestinal organoids. Specific results are as follows: Figure 4 As shown. Figure 4 The immunohistochemical staining results are shown, with clear brown-yellow positive expression of the LYZ marker, confirming the presence of Paneth cells in the intestinal organoids and verifying that capillary-cultured organoids possess functional characteristics similar to real tissues. This figure provides crucial evidence for the reliability of this method.

[0027] The capillary culture method of this invention, employing a dynamic culture system, significantly overcomes the shortcomings of traditional well plates. It allows for more balanced and consistent nutrient uptake by organoids, facilitates the timely removal of metabolic waste, and enables better prediction of drug response and therapeutic changes in vivo. This method is simple, easy to operate, and inexpensive. This dynamic capillary culture has also greatly promoted the rise of novel dynamic organoid culture models.

[0028] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method based on capillary culture organoids, characterized by: The cell mass to be cultured is mixed with the diluted Matrigel to form a suspension, which is injected into the capillary tube by capillary action; then the capillary tube is placed in the cell incubator, and after the suspension in the capillary tube is naturally solidified, the capillary tube is taken out of the cell incubator, the complete organoid culture medium is filled above the solidified suspension, and then the capillary tube is hung on the support and placed in the cell incubator for culture. The complete organoid culture medium continuously infiltrates, uniformly spreads and flows into the Matrigel-embedded suspension. As the culture time increases and the organoids grow, the organoids continuously consume the nutrients in the complete organoid culture medium. By timely updating the complete organoid culture medium, the dynamic and stable growth of the organoids in the capillary tube is realized.

2. The capillary culture organoid-based method of claim 1, wherein: The complete organoid culture medium is updated by injecting new complete organoid culture medium into the capillary tube at intervals.

3. The capillary culture organoid-based method of claim 1, wherein: The capillary tube is a "┌" type capillary tube with a horizontal section and a vertical section. The end of the vertical section is injected with the suspension by capillary action. After natural solidification in the cell incubator, the complete organoid culture medium is injected into the cavity above the suspension and the horizontal section through the connecting hose in the syringe, and then the capillary tube is hung on the support and placed in the cell incubator for continuous culture.

4. A device for culturing organoids based on capillaries for carrying out the method according to any one of claims 1 to 3, characterized in that The capillary tube is hung on the support, and the support is placed in the cell incubator.

5. The apparatus of claim 4, wherein, The material of the capillary tube is one of glass, quartz and polymer.

6. The apparatus of claim 5, wherein, The shape of the capillary tube is round or square.

7. The apparatus of claim 5, wherein, The inner diameter of the capillary tube is 0.9-1.1mm.

8. The apparatus of claim 4, wherein, The support is composed of two identical base columns and an upper plate. The two base columns support the upper plate, and the upper plate is provided with a plurality of holes or clamping structures for fixing the capillary tube.

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