Organ-like core-shell microspheres as well as preparation method and application thereof

Abstract

The invention relates to an organ-like core-shell microsphere as well as a preparation method and application thereof. The preparation method comprises the steps that 1, raw materials are prepared, specifically, shell raw materials and inner core raw materials are prepared, the shell raw materials comprise a photoinitiator and methacrylated hyaluronic acid (HAMA), and the inner core raw materials comprise organoid precursor cell suspension and matrigel; 2) molding: extruding the dispersion phase of the shell raw material wrapping the core raw material to a continuous phase by using a micro-fluidic chip and a high-precision injection pump, cutting the dispersion phase into liquid drops by the continuous phase, and performing illumination curing on the liquid drops to form core-shell microspheres; and (3) incubating, namely culturing the core-shell microspheres in a culture medium, so that the organoid precursor cells in the inner core are developed into organoids. According to the preparation method, rapid forming and curing of the organ-like precursor cells carried by the matrigel are achieved, so that the time of the organ-like precursor cells staying in the oil phase is shortened, the cells can obtain oxygen and nutrient substances, and the cell activity is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of organoid culture, in particular to an organoid core-shell microsphere, a preparation method and application thereof. BACKGROUND

[0002] Organoids are micro-organ models formed by three-dimensional in vitro culture technology from organoid precursor cells (which can be stem cells, tumor tissue cells or healthy tissue cells). They can simulate the structure and function of real organs and are widely used in disease research, drug screening and regenerative medicine.

[0003] The most commonly used culture matrix for organoid technology is Matrigel. Stem cells are wrapped in Matrigel to provide a three-dimensional, nutrient-rich microenvironment to promote the formation of organoids. However, the current organoid technology has limitations such as high reagent cost, long culture time (several weeks), low automation and standardization, and difficulty in immune co-culture, which limits its widespread application.

[0004] In view of the above limitations, in recent years, organoid technology has been combined with microsphere manufacturing technology, which has the following unique advantages: (1) Organoid microsphere technology can reduce the consumption of Matrigel and culture medium. For example: the volume of a single traditional organoid Matrigel dome-shaped droplet is usually 10-40 μL, and if it is cultured in a 24-well plate, at least 500 μL of culture medium is required. However, the volume of a 500 μm diameter organoid microsphere is only 65 nL, which is 1 / 200 of the volume of a traditional organoid droplet. Therefore, less Matrigel is consumed, and 384-well plates can be used for culture, requiring only 50 μL of culture medium, greatly reducing the amount of expensive reagents and the cost of organoid culture.

[0005] (2) The preparation of organoid microspheres relies on automated equipment, improving automation and reducing errors caused by manual operation. The experimental parameters are adjustable, the microspheres are uniform in size, and the diameter is controllable.

[0006] (3) Traditional organoid droplets have a large volume, and immune cells are difficult to penetrate the entire layer, so the immune co-culture effect is not good. However, the small volume of organoid microspheres helps immune cells to penetrate, enabling immune co-culture-related experiments.

[0007] Some prior art proposes a method for manufacturing organoid Matrigel microspheres, which is based on droplet microfluidic technology, prepares organoid precursor cell / Matrigel blend microspheres, and adopts a temperature solidification strategy, i.e. incubation at 37℃ to solidify Matrigel, but the whole process still takes about 30 minutes. It can be seen that this forming method is low in efficiency and time-consuming, and when the organoid precursor cell / Matrigel blend is still in the oil phase during temperature solidification, the organoid precursor cells cannot be in contact with the outside air and sufficient nutrients for a long time, which affects the survival rate of the organoid precursor cells and is not conducive to the formation of subsequent organoids. Moreover, the mechanical properties of Matrigel itself are still poor after solidification, and the microsphere structure is easily broken during subsequent culture and liquid exchange operations, resulting in the leakage of organoids and the loss of suitable growth environment. SUMMARY

[0008] The present application aims to disclose an organoid core-shell microsphere and a preparation method and application thereof, to solve one or more technical problems in the prior art and provide at least one beneficial option or create conditions.

[0009] The first aspect of the present application is to provide a preparation method of an organoid core-shell microsphere.

[0010] The second aspect of the present application is to provide the organoid core-shell microsphere.

[0011] The third aspect of the present application is to provide the application direction of the organoid core-shell microsphere.

[0012] The preparation method of the first aspect of the present application comprises the following steps: 1) preparing raw materials: preparing shell raw materials and core raw materials, the shell raw materials containing a photoinitiator and a photosensitive hydrogel material, the core raw materials including an organoid precursor cell suspension and Matrigel, and the photosensitive hydrogel material being selected from methacrylated hyaluronic acid (HAMA), methacrylated sodium alginate or polyethylene glycol diacrylate; 2) forming: using a microfluidic chip and a high-precision syringe pump to extrude the dispersed phase of the core raw materials wrapped by the shell raw materials to the continuous phase, the dispersed phase being cut into droplets by the continuous phase, and the droplets being solidified by light to form core-shell microspheres; 3) incubation: culturing the core-shell microspheres in a culture medium to develop the organoid precursor cells in the core into organoids.

[0013] The preparation method is based on photocrosslinking solidification technology and core-shell microsphere technology, and optimizes the molding scheme of organoid core-shell microspheres. The organoid precursor cell suspension and Matrigel mixture are used as the inner core raw material, a water-soluble hydrogel material with rapid solidification, excellent mechanical properties and good biocompatibility is selected as the outer shell raw material to wrap the inner core raw material, and the core-shell microspheres are formed by rapid solidification of the outer shell. The method can significantly improve the solidification efficiency of microsphere preparation and the mechanical properties of the microspheres.

[0014] In some application embodiments of the first aspect of the present application, the concentration of organoid precursor cells in the organoid precursor cell suspension is 2×10 5 ~2×10 7 Preferably, the organoid precursor cells include primary cells and non-primary cell lines. Primary cells are closer to the physiological state, but are complex and costly to operate, suitable for individualization or high simulation research. Non-primary cell lines are easy to operate and standardized, but may lose part of the biological properties, suitable for mechanism research or large-scale application. However, since the core-shell microspheres prepared by the present preparation method use Matrigel mixed organoid precursor cell suspension as the culture environment, the quality of the obtained organoids will not be reduced regardless of which one is selected as the organoid precursor cell.

[0015] In some application embodiments of the first aspect of the present application, the volume ratio of the Matrigel to the organoid precursor cell suspension in the inner core raw material is (1-9):1; preferably, the volume ratio of the Matrigel to the organoid precursor cell suspension is 2:1.

[0016] In some application embodiments of the first aspect of the present application, the concentration of the photosensitive hydrogel material is in the range of 0.1-15% (w / v). Preferably, when the concentration of the photosensitive hydrogel material is in the range of 2-4% (w / v), the flowability of the outer shell raw material during preparation will be better, so that the outer shell after solidification is more regular.

[0017] In some application embodiments of the first aspect of the present application, the photo initiator in the outer shell raw material is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (photo initiator LAP), and the concentration is 0.1-1%; preferably, the concentration of the photo initiator LAP in the outer shell raw material is 0.25%.

[0018] In some application embodiments of the first aspect of the present application, the continuous phase is 0.05-5% (v / v) Span 80 corn oil; preferably, the continuous phase is 1% (v / v) Span 80 corn oil.

[0019] In some application embodiments of the first aspect of the present application, the microfluidic chip is a glass-based chip assembled by coaxial capillary tubes, or a polydimethylsiloxane chip with multiple flow channels.

[0020] In some embodiments of the first aspect of the application, the preparation of the raw material and / or the forming step are carried out at 2-8℃. In addition to HAMA, common photosensitive hydrogel materials also include methacrylated gelatin, methacrylated sodium alginate, methacrylated chitosan, methacrylated chondroitin sulfate, and polyethylene glycol diacrylate. However, since the matrix glue has better flowability at 2-8℃, it is necessary to select HAMA as the shell raw material which also has better flowability at the same temperature, so as to make the preparation of the core-shell microspheres easy to control.

[0021] The core-shell microspheres of the second aspect of the application have a diameter of 200-1000 μm, and comprise an inner core and an outer shell. The inner core contains matrix glue and organoids, and the outer shell is formed by light-cured crosslinking of HAMA, which wraps the inner core.

[0022] Since the core-shell microspheres use HAMA as the main material of the outer shell, rapid solidification and forming can be achieved in 1-5 seconds in a small diameter range, avoiding the problem that organoid precursor cells stay in the continuous phase for a long time and cannot contact the outside air. Moreover, since HAMA has degradable properties, the shell can be digested by enzymatic digestion in subsequent research and use, thereby releasing the internal organoids without affecting the subsequent experimental characterization of cell biology.

[0023] In some embodiments of the second aspect of the application, the concentration of the matrix glue in the inner core is 50-90%.

[0024] The application of the third aspect of the application refers to the use of the core-shell microspheres for constructing physiological and pathological models, studying the formation, proliferation, differentiation, and apoptosis of organoids, co-culturing with other cells, including vascular endothelial cells, fibroblasts, and immune cells, to study their interactions, or for studying the effects of drugs or other active molecules on them to evaluate the effects of drugs.

[0025] Compared with the existing technology, the application has the following advantages: (1) On the premise of retaining the advantages of small volume, uniform size, automated preparation, and immune co-culture of microspheres, the core-shell microsphere design and light-curing strategy are used to rapidly form and solidify the matrix glue carrying organoid precursor cells for the first time. The traditional 20-30 minutes is shortened to 1-5 seconds, improving the solidification efficiency and shortening the preparation time.

[0026] (2) The mechanical properties of the shell are better than those of the solidified matrix glue, which can effectively reduce the risk of microsphere damage caused by liquid exchange and other operations during culture, and ensure the integrity of the microsphere morphology and structure.

[0027] (3) The shell has degradable properties, and the shell can be dissolved using corresponding digestive enzymes to release the organoids without affecting subsequent experimental characterization of cell biology. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is a schematic diagram of a preparation method of the organoid core-shell microspheres. Figure 2 is a photo of the core-shell microspheres prepared in Example 2 on the day of preparation; Figure 3 is a photo of the core-shell microspheres prepared in Example 2 seven days after preparation; Figure 4 is a photo of the organoid core-shell microspheres before and after enzymatic digestion in Example 3; Figure 5 is a photo of the core-shell microspheres prepared in Example 4 seven days after culture; Figure 6 is a photo of the core-shell microspheres prepared in Example 5 seven days after culture; Figure 7 is a dose-response curve of the drug sensitivity test of the core-shell microspheres prepared in Example 5. DETAILED DESCRIPTION

[0029] The following examples further illustrate the present application, but should not be construed as limiting the present application. Modifications and substitutions can be made to the methods, steps or conditions of the present application without departing from the spirit and scope of the present application.

[0030] Unless specifically indicated otherwise, the technical means used in the examples are conventional means known to those skilled in the art.

[0031] The molecular biology test methods not specifically described in the following examples are performed according to the Molecular Cloning Laboratory Guide (3rd edition) or according to the reagent kit and product instructions; the biological materials of the reagent kit, if not specifically indicated, can be obtained from commercial channels.

[0032] Example 1: Preparation method of organoid core-shell microspheres The preparation schematic diagram of the organoid core-shell microspheres is shown in Figure 1 .

[0033] The organoid precursor cells and the Matrigel mixture serve as the inner core material, the HAMA solution and the photoinitiator serve as the outer shell material, and the corn oil serves as the continuous phase. The inner core material and the outer shell material are injected into channel 1 and channel 2 respectively through high-precision syringe pumps, and at the outlet, the dispersed phase of the outer shell material wrapping the inner core material is formed and then enters channel 3 filled with the flowing continuous phase. The dispersed phase entering channel 3 is intercepted by the continuous phase to form droplets of the outer shell material wrapping the inner core material. After the droplets pass through the irradiation of UV lamp 5, the outer shell material is rapidly solidified, and the droplets are converted into core-shell microspheres 6.

[0034] To ensure the fluidity of the inner core material and the outer shell material, the temperature in the temperature control area 4 is kept at 2-8°C.

[0035] The prepared core-shell microspheres 6 are collected and placed in the corresponding organoid culture medium for cultivation in a carbon dioxide incubator, so that the organoid precursor cells develop into organoids, and the organoid core-shell microspheres are obtained.

[0036] Example 2: Preparation of endometrial cancer organoid core-shell microspheres 1) Preparation of raw materials: the outer shell material contains 0.25% photoinitiator LAP and 2% HAMA; the inner core material includes a suspension of endometrial cancer cells with a concentration of about 5×10 6 6 cells / mL and Matrigel, and the volume ratio of the suspension to Matrigel is 1:2, and the operation is performed on ice; 2) Molding: corn oil containing 1% (v / v) Span 80 is selected as the continuous phase. 1 mL of the inner core material, 1 mL of the outer shell material, and 50 mL of the continuous phase are filled into syringes of high-precision syringe pumps, which are connected to the corresponding extrusion channels, and the syringes are connected to the microfluidic chip through hoses. The outlet of the microfluidic chip is stretched into the PBS solution through a transparent silicone hose. The syringe pump loaded with the inner core material and the microfluidic chip are cooled to 2-8°C by covering them with crushed ice. After the preparation is completed, the high-precision syringe pumps are started, and when the flow rates of the inner core material, the outer shell material, and the continuous phase are stabilized at 5 μL / min, 5 μL / min, and 500 μL / min respectively, it can be observed that the dispersed phase in the microfluidic chip is intercepted by the continuous phase to form spherical droplets with uniform size. The laser generator is turned on, and the spherical droplets in the continuous phase are irradiated with blue light with a wavelength of 405 nm, so that they are solidified into core-shell microspheres within 1-5 seconds.

[0037] 3) Incubation: After the preparation is completed, the laser generator and the high-precision syringe pumps are turned off, the collected core-shell microspheres and the PBS solution are centrifuged at 500 rpm for 2 minutes to remove the upper oil phase, then washed with PBS for 3 times, and transferred to the endometrial cancer culture medium and cultured in the cell culture incubator, and the culture medium is replaced every 3-4 days.

[0038] The prepared core-shell microspheres can withstand centrifugal treatment without breaking. The core-shell microspheres prepared on the same day can be observed under a microscope as shown in FIG. 6A, and it can be seen that the microspheres are uniform in size, with a diameter of about 400 μm, and the cells in the core are distributed in a dispersed or small clump form. Figure 2 After 7 days of culture, the volume of the cell growth clumps can be observed to have increased significantly, forming organoids, and the microsphere structure is maintained without breaking, i.e., the endometrial cancer organoid core-shell microspheres are obtained. Figure 3

[0039] Example 3: Preparation of colorectal cancer organoid core-shell microspheres The organoids have a wide range of research applications, such as for slicing to observe the tissue structure, DNA or RNA sequencing of cells in the organoids, determination of cell number viability, or protein extraction, etc. Before performing these application operations, the organoid core-shell microspheres need to be opened to release the organoids therein for the next step operation.

[0040] This example takes the colorectal cancer organoid core-shell microspheres as an example to demonstrate the method for extracting the organoids from the core-shell microspheres.

[0041] The preparation process of the colorectal cancer organoid core-shell microspheres is similar to the preparation method described in Example 2, except that the colorectal cancer cells are used instead of the endometrial cancer cells in this example, and the culture medium used in the culture process is suitable for the culture of colorectal cancer organoids.

[0042] After 5 days of preparation of the above-mentioned core-shell microspheres, hyaluronidase with a concentration of 10 U / mL is added to the culture solution, mixed and blown thoroughly, and placed in a culture box for digestion. The situation is observed every 5 minutes, mixed and blown thoroughly, and the degradation process is completed in about 15-30 minutes. Microscopic observation shows that the left image is before the degradation of the shell, and the right image is after the degradation of the shell, as shown in FIG. 7. Figure 4

[0043] Example 4: Preparation of mouse lung organoid core-shell microspheres The preparation process is similar to the preparation method described in Example 2, except that the normal lung cells of mice are used to prepare the organoid precursor cell suspension in this example, and the culture medium used is suitable for the culture of mouse lung organoids, and the formed organoid core-shell microspheres are mouse lung organoid core-shell microspheres.

[0044] The photograph of the mouse lung organoid core-shell microspheres after 7 days of culture is shown in FIG. 8, and it can be observed that the organoid precursor cells in the microspheres self-assemble to develop organoid structures with a diameter of more than 100 microns. Figure 5

[0045] Example 5: Preparation of kidney cancer organoid core-shell microspheres​​​ The preparation process is similar to the preparation method described in Example 2, except that the kidney cancer cell is used to prepare the organoid precursor cell suspension, the culture used is the culture medium suitable for kidney cancer organoids, and the formed organoid core-shell microspheres are kidney cancer organoid core-shell microspheres.

[0046] After the core-shell microspheres are prepared and cultured, they are transferred to a 384-well plate 4 days later, with 40 μL of culture medium per well, containing 10-30 kidney cancer organoid core-shell microspheres. Then, drug-containing culture medium with drug concentrations of 50 μmol / L, 10 μmol / L, 2 μmol / L, 0.4 μmol / L, 0.08 μmol / L, and 0 μmol / L is added, and the drugs include axitinib, lenvatinib, ruboxatil, sorafenib, and sunitinib. After 4 days of drug action, 40 μL of luminescence-based organoid viability detection reagent (CellTiter Glo) is added per well, and after incubation at room temperature for 30 minutes, the chemiluminescence value is detected using a microplate reader, and statistical analysis is performed to obtain the drug sensitivity test results.

[0047] The kidney cancer organoid core-shell microspheres negative control (drug-free group 9) during the drug test is shown in Figure 6 It can be seen that organoids have been formed, indicating that the drug does indeed act on the organoids rather than single cells, which meets the definition of organoid killing. The dose-response curve of the drug sensitivity detection is shown in Figure 7 The horizontal axis is the logarithmic value of the drug concentration, and the results show that the curves of sorafenib and sunitinib tend to decrease and cross the cell activity = 50% boundary, so it can be judged that the kidney cancer organoids may be sensitive to sorafenib and sunitinib. In contrast, the curves of axitinib, lenvatinib, and ruboxatil show no downward trend or only a slight decrease, indicating that the kidney cancer organoids are not sensitive to axitinib, lenvatinib, and ruboxatil.

[0048] It is obvious to those skilled in the art that the present application is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or essential characteristics of the present application. Therefore, the embodiments should be considered as exemplary and non-limiting, and the scope of the present application is defined by the appended claims rather than the above description, and all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present application.

Claims

1. An organoid core-shell microsphere, characterized in that, The diameter of the organoid core-shell microsphere is 200-1000 μm, which comprises an inner core and a shell, the inner core contains a Matrigel and an organoid, the shell is formed by photo-crosslinking of a photosensitive hydrogel material, the shell wraps the inner core, and the photosensitive hydrogel material is selected from methacrylated hyaluronic acid, methacrylated sodium alginate or polyethylene glycol diacrylate.

2. The organoid core-shell microsphere of claim 1, wherein, The concentration of the Matrigel in the inner core is 50-90%.

3. The method of claim 1 or 2, wherein the organoid core-shell microspheres are prepared by, The method comprises the following steps: 1) preparing raw materials: preparing a shell raw material and an inner core raw material, the shell raw material contains a photoinitiator and methacrylated hyaluronic acid, and the inner core raw material comprises an organoid precursor cell suspension and a Matrigel; 2) forming: using a microfluidic chip and a high-precision syringe pump to extrude the shell raw material to wrap the dispersed phase of the inner core raw material into a continuous phase, the dispersed phase is cut into droplets by the continuous phase, and the droplets are cured by light to form core-shell microspheres; 3) incubation: placing the core-shell microspheres in a culture medium for culture, so that the organoid precursor cells in the inner core develop into organoids.

4. The preparation method according to claim 3, characterized in that, The concentration of organoid precursor cells in the organoid precursor cell suspension is 2 x 10 5 ~2 x 10 7 cells / mL; preferably, the organoid precursor cells comprise primary cells and non-primary cell lines.

5. The preparation method according to claim 3, characterized in that, In the inner core raw material, the volume ratio of the Matrigel to the organoid precursor cell suspension is (1-9):1; preferably, the volume ratio of the Matrigel to the organoid precursor cell suspension is 2:

1.

6. The preparation method according to claim 3, characterized in that, The concentration of the methacrylated hyaluronic acid ranges from 0.1% to 15% (w / v); preferably, the concentration ranges from 2% to 4%.

7. The preparation method according to claim 6, characterized in that, The photoinitiator in the shell raw material is lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and the concentration is 0.1-1%; preferably, the concentration of the lithium phenyl-2,4,6-trimethylbenzoylphosphinate in the shell raw material is 0.25%.

8. The preparation method according to claim 3, characterized in that, The continuous phase is corn oil containing 0.05-5% (v / v) Span 80; preferably, the continuous phase is corn oil containing 1% (v / v) Span 80.

9. The preparation method according to claim 3, characterized in that, The microfluidic chip is a glass-based chip assembled by coaxial capillary tubes, or a polydimethylsiloxane chip with multiple flow channels.

10. The preparation method according to claim 3, characterized in that, The preparation of raw materials and / or the forming step are both carried out at 2-8°C.

11. The organoid core-shell microsphere of claim 1 or 2 for use in physiological and pathological model construction and drug reaction evaluation.

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