A porous plate for culturing cell aggregates and its preparation and use methods

Abstract

The present invention relates to a porous plate for culturing cell aggregates, and a preparation and use method thereof. The porous plate includes: a plate body, the material forming the plate body being a hydrogel material prepared from a hydrophilic natural polymer, and the hydrogel material being capable of undergoing gel-sol transformation; a pore body, the pore body being provided on at least one surface of the plate body. The technical solution of the present invention can promote the spontaneous aggregation of cells, facilitate the one-time preparation of a large number of cell aggregates with uniform size, and enable rapid and efficient collection.

Description

Technical Field

[0001] The present invention relates to the technical field of cell body culture, and in particular to a porous plate for culturing cell aggregates and a method for manufacturing and using the same. Background Art

[0002] A cell aggregate is a form of cell existence formed by the aggregation of multiple single cells. Generally, it is formed due to the need to resist the influence of adverse external environments. When cells cannot form a tight interaction with materials, cells will also aggregate to form cell aggregates.

[0003] The interaction between cells in cell aggregates is enhanced, and cells secrete a large amount of extracellular matrix and proteins, greatly enhancing the ability of aggregates to resist external influences. At the same time, the aggregated cells can grow in three dimensions, which is very close to the cell growth environment in real tissues, facilitating further research on the tissue growth process or the disease occurrence process. In addition, studies have shown that cell aggregates can fuse with each other to form larger cell aggregates, which provides the possibility for constructing tissues or organs in vitro.

[0004] How to efficiently prepare cell aggregates has been a research hotspot in recent years. Traditional preparation methods mainly include methods such as rotary culture, constructing anti-adhesion matrices, and external force induction. However, these methods have disadvantages such as complex operations, high costs, uncontrollable sizes of the formed cell aggregates, and low cell utilization rates, and cannot achieve efficient preparation. With the development of technologies such as lithography and microfluidics, some studies use porous arrays with special shapes to prepare cell aggregates, which have high cell utilization rates and uniform cell aggregate sizes, enabling efficient preparation. However, after the cell aggregation process ends, the cell aggregates remain at the bottom of the array holes and can only be collected by means of multiple pipetting. During the collection process, external forces will tear the cells or extracellular matrix in the aggregates, and at the same time, the collection process is time-consuming and laborious. Currently, relatively mature products for preparing cell aggregates on the market, such as the Corning Ultra-Low Attachment Well Plate series products, require the use of a pipette to collect the aggregates in each well sequentially during the collection process, which is time-consuming. Developing a product that can be conveniently collected and can efficiently prepare cell aggregates is the key to promoting the development of related fields. Summary of the Invention

[0005] To solve the problems of complex operation and difficulty in collection in the prior art when preparing and collecting cell aggregates, the present invention provides a porous plate for culturing cell aggregates that is convenient to operate and easy to collect, as well as its preparation and use methods. This porous plate can culture a large number of cell aggregates with uniform size at one time. The cell aggregates with uniform size prepared by this method have unique advantages in the application of three-dimensional tissue printing because they can fit the printing channels, and some microtissue constructs based on the kenzan needle array can only use cell aggregates with uniform size.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions:

[0007] 1. A porous plate for culturing cell aggregates, comprising:

[0008] A plate body, the material forming the plate body is a hydrogel material prepared from a hydrophilic natural polymer, and the hydrogel material can undergo gel-sol transformation;

[0009] Hole bodies, the hole bodies are arranged on at least one surface of the plate body.

[0010] 2. The porous plate according to item 1, wherein the hydrogel material is formed by connecting the polymer chains of the hydrophilic natural polymer through dynamic covalent bonds.

[0011] 3. The porous plate according to item 2, wherein the hydrogel material is formed by crosslinking the hydrophilic natural polymer using a crosslinking agent containing dynamic covalent bonds.

[0012] 4. The porous plate according to item 2, functional groups are introduced into the hydrophilic natural polymer, and the hydrophilic natural polymer forms the hydrogel material through the formation of dynamic covalent bonds between the introduced functional groups.

[0013] 5. The porous plate according to item 2, wherein the dynamic covalent bonds include one or more of Schiff base bonds, disulfide bonds, borate ester bonds, and acylhydrazone bonds.

[0014] 6. The porous plate according to item 1, functional groups are introduced into the hydrophilic natural polymer, and the hydrophilic natural polymer forms the hydrogel material through the formation of non-covalent bonds between the introduced functional groups.

[0015] 7. The porous plate according to item 6, the formation of the non-covalent bonds is achieved through one or more of host-guest interactions, reversible hydrogen bonds, metal coordination, hydrophobic interactions, van der Waals forces, and π-π interactions.

[0016] 8. The porous plate according to item 1,

[0017] A plurality of the pore body arrays are arranged on the plate body, specifically, for example, pore bodies arranged in x rows and y columns (2 ≤ x ≤ 50, 2 ≤ y ≤ 50, x and y are integers) can be provided.

[0018] The diameter of the pore body is 200 - 1000 μm, the depth of the pore body is 200 - 1000 μm, and the distance between adjacent two pore bodies is 50 - 200 μm.

[0019] 9. The porous plate according to item 1, wherein the hydrophilic natural polymer is selected from one or a combination of two or more materials of hyaluronic acid, polyglutamic acid, chitosan, alginic acid, and heparin; preferably hyaluronic acid with a molecular weight of 40 kDa - 400 kDa.

[0020] 10. The porous plate according to item 3, wherein the crosslinking agent is cystamine dihydrochloride.

[0021] 11. The porous plate according to item 3, wherein the hydrophilic natural polymer reacts with an activator before crosslinking, and the activator activates the carboxyl groups in the hydrophilic natural polymer.

[0022] 12. The porous plate according to item 11, wherein the activator is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

[0023] 13. A preparation method of a porous plate for culturing cell aggregates, comprising:

[0024] Dissolution step: Dissolve the hydrophilic natural polymer to prepare a polymer solution.

[0025] Crosslinking and molding step: Add a crosslinking agent to the polymer solution and cure it into a hydrogel on a mold. Multiple columns are provided in the mold. The crosslinking agent contains one or two or more chemical bonds of Schiff base bond, disulfide bond, borate ester bond, and hydrazone bond. Preferably, the crosslinking agent contains a disulfide bond.

[0026] 14. The preparation method according to item 13, wherein the hydrophilic natural polymer is selected from one or a combination of two or more materials of hyaluronic acid, polyglutamic acid, chitosan, alginic acid, and heparin, and preferably hyaluronic acid with a molecular weight of 40 kDa - 400 kDa.

[0027] 15. The preparation method according to item 13, further comprising an activation step before the crosslinking and molding step;

[0028] The activation step is to add an activator to the polymer solution;

[0029] The activator activates the carboxyl groups in the hydrophilic natural polymer.

[0030] 16. The preparation method as described in item 15, wherein the activator is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide.

[0031] 17. The preparation method as described in item 13, wherein the crosslinking agent is cystamine dihydrochloride.

[0032] 18. The preparation method as described in item 13, wherein the column in the mold is formed by lithography of silica or 3D printing; multiple columns are arranged in an array; the diameter of the column is 200 - 1000 μm, the height of the column is 200 - 1000 μm, and the distance between adjacent two columns is 50 - 200 μm.

[0033] 19. The preparation method as described in item 13 further includes a dialysis step, which is to perform gradient dialysis on the hydrogel using deionized water.

[0034] 20. A preparation method of a porous plate for culturing cell aggregates, comprising:

[0035] Dissolving a hydrophilic natural polymer introduced with functional groups to prepare a polymer solution;

[0036] The polymer solution forms a hydrogel by forming dynamic covalent bonds between the introduced functional groups, thereby preparing the porous plate.

[0037] 21. A preparation method of a porous plate for culturing cell aggregates, comprising:

[0038] Dissolving a hydrophilic natural polymer introduced with functional groups to prepare a polymer solution;

[0039] The polymer solution forms a hydrogel by forming non-covalent bonds between the introduced functional groups, thereby preparing the porous plate.

[0040] 22. The usage method of the porous plate as described in item 1, comprising:

[0041] A sterilization step of sterilizing the porous plate;

[0042] An inoculation and culturing step of inoculating cells in the pore bodies of the porous plate for culturing;

[0043] A decomposition step of obtaining a cell aggregate suspension by gel-sol transformation of the porous plate after forming cell aggregates;

[0044] A collection step of separating and collecting cell aggregates.

[0045] 23. The usage method of the porous plate as described in item 22, wherein the sterilization step is to soak the porous plate with alcohol and then replace it with PBS.

[0046] 24. The method for using the porous plate according to item 22, wherein in the collection step, the cell aggregate suspension is centrifuged to separate the cell aggregates.

[0047] 25. The method for using the porous plate according to item 22, when the hydrogel material is formed by connecting the polymer chains of the hydrophilic natural polymer through disulfide bonds in dynamic covalent bonds, the decomposition step is to add a reducing agent containing a thiol group to the porous plate after forming the cell aggregates; the reducing agent is preferably glutathione.

[0048] 26. The method for using the porous plate according to item 25, wherein the concentration of the glutathione is 10 - 30 mmol / L, preferably 13 - 17 mmol / L.

[0049] The material used for the porous plate of the present invention is a gel material that can undergo gel-sol transformation and is prepared from natural polymer materials. The hydrophilicity of the natural polymer is utilized to promote the spontaneous aggregation of cells; moreover, the porous plate can prepare a large number of cell aggregates with uniform sizes at one time, and the preparation process is convenient and efficient. The column array formed by photolithographic silica molding or 3D printing molding has high precision and is convenient for adjusting the column size according to requirements; more importantly, during the collection process of the cell aggregates in the porous plate of the present invention, through gel-sol transformation, a mixed solution of cell aggregates and natural polymer can be obtained, and the cell aggregates with uniform sizes can be collected by centrifugation, which has unique advantages. Description of the Drawings

[0050] Figure 1 : Microscopic photograph of cell aggregates in Example 1;

[0051] Figure 2 : Schematic structural diagram of the porous plate;

[0052] Figure 3 : In a porous plate Figure 2 Schematic diagram of the A - A' cross-section;

[0053] Figure 4 : And Figure 3 Schematic structural diagram of the mold corresponding to the porous plate;

[0054] Figure 5 : In another porous plate Figure 2 Schematic diagram of the A - A' cross-section;

[0055] Figure 6 : And Figure 5 Schematic structural diagram of the mold corresponding to the porous plate;

[0056] Figure 7 : Microscopic photograph of cell aggregates in Comparative Example 2.

[0057] Reference numerals: 1, porous plate; 1-1, plate body; 1-2, pore body; 2, mold; 2-1, column body. Detailed implementation manners

[0058] The following describes exemplary embodiments of the present application, including various details of the embodiments of the present application to facilitate understanding. It should be considered merely exemplary. Therefore, those of ordinary skill in the art should recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. Similarly, for clarity and conciseness, descriptions of well-known functions and structures are omitted in the following description.

[0059] This embodiment provides a porous plate 1 for culturing cell aggregates, as Figures 1 to 6 shown, including:

[0060] A plate body 1-1, and the material forming the plate body is a hydrogel material prepared from a hydrophilic natural polymer, and the hydrogel material can undergo a gel-sol transition;

[0061] Pore bodies 1-2, and a plurality of pore bodies are provided on at least one surface of the plate body.

[0062] A hydrophilic natural polymer material refers to a natural polymer that can dissolve or swell in water to form a solution or dispersion.

[0063] The hydrophilic natural polymer material can form a stable hydration layer with water molecules. When the porous plate prepared from it is used for culturing cell aggregates, the hydration layer hinders the interaction between cells and the natural polymer, weakening the interaction between cells and the matrix and enhancing the interaction between cells, causing the cells to spontaneously form aggregates. Therefore, the porous plate of this embodiment is conducive to the formation of cell aggregates.

[0064] Gel-sol transition refers to the transformation of a quasi-solid-state hydrogel into a sol or solution.

[0065] The hydrogel material of this embodiment can undergo a gel-sol transition. Then, cell aggregates are cultured in the gel state. After that, the gel material can be transformed into a sol or solution through the gel-sol transition, obtaining a mixed solution of cell aggregates and natural polymer. Centrifugation can be used to collect cell aggregates with uniform size. Compared with the prior art, it is more convenient to collect and more efficient in preparing cell aggregates.

[0066] In addition, it should be noted that Figures 2 to 6 merely for schematic purposes, the porous plate of the present application can be such that Figure 3 the bottom of the pore body 1-2 shown in is a flat bottom, or it can be Figure 5The bottom of the shown hole body 1-2 is a spherical round bottom or other shapes, and the number of hole bodies 1-2 can also be adjusted and changed according to specific needs.

[0067] In an embodiment of the porous plate, the hydrogel material is formed by connecting the polymer chains of the hydrophilic natural polymer through dynamic covalent bonds.

[0068] Dynamic covalent bonds refer to a class of covalent bonds that can exchange or transform between molecules and can be reversibly broken or generated under certain conditions.

[0069] This application presents the realization of the gel-sol transition of the hydrogel material by preparing the hydrogel material through dynamic covalent bonds.

[0070] In an embodiment of the porous plate, the hydrogel material is formed by crosslinking the hydrophilic natural polymer using a crosslinking agent containing dynamic covalent bonds.

[0071] The hydrophilic natural polymer material contains a large number of hydroxyl and carboxyl groups. The active groups such as amino and epoxy groups in the crosslinking agent react with the hydroxyl and carboxyl groups in the hydrophilic natural polymer material to achieve crosslinking and obtain the hydrogel material, thereby preparing a porous plate for culturing aggregates. The porous plate realizes the gel-sol transition through the breaking of the dynamic covalent bonds in the crosslinking agent.

[0072] In an embodiment of the porous plate, functional groups are introduced into the hydrophilic natural polymer, and the hydrophilic natural polymer forms the hydrogel material through the formation of dynamic covalent bonds between the introduced functional groups.

[0073] Specifically, for example, by introducing amino and aldehyde groups into the hydrophilic natural polymer hyaluronic acid respectively, after the obtained hydrophilic natural polymer is prepared into an aqueous solution, the pH can be adjusted to weakly alkaline to make the amino and aldehyde groups undergo a Schiff base reaction to form an imine bond (Schiff base bond) to obtain the hydrogel material.

[0074] In an embodiment of the porous plate, the dynamic covalent bonds include one or more of Schiff base bonds, disulfide bonds, borate ester bonds, and acylhydrazone bonds.

[0075] This embodiment lists some dynamic covalent bonds. The hydrogel material is formed by crosslinking a hydrophilic natural polymer with a crosslinking agent containing Schiff base bonds, disulfide bonds, borate ester bonds or hydrazone bonds, or by forming the above-mentioned dynamic covalent bonds between specific functional groups introduced, and the porous plate prepared therefrom can be used for culturing cell aggregates. Subsequently, the above-mentioned dynamic covalent bonds can be broken by adding a corresponding reducing agent, changing the pH, using special wavelength light irradiation, etc. to achieve the gel-sol transition and convert the hydrogel material into a sol, thus obtaining a mixed solution of cell aggregates and natural polymer, and the cell aggregates with uniform size can be collected by centrifugation.

[0076] It should be noted that the reducing agent in this application refers to a reagent that can break dynamic covalent bonds.

[0077] In an embodiment of a porous plate, functional groups are introduced into the hydrophilic natural polymer, and the hydrophilic natural polymer forms the hydrogel material by forming non-covalent bonds between the introduced functional groups.

[0078] Non-covalent bonds refer to chemical systems in supramolecular chemistry that form molecular assemblies or aggregation structures through interactions other than forming covalent bonds, including host-guest interactions, reversible hydrogen bonds, metal coordination, hydrophobic interactions, van der Waals forces, and π-π interactions, etc. Non-covalent bonds form assemblies or complexes only by intermolecular or ionic interactions, and their stability is poor, and they can usually respond to environmental stimuli (such as pH, light, redox properties, and temperature, etc.) and reversibly form or disintegrate. Therefore, by utilizing the self-composition and structural characteristics of natural hydrophilic polymers or introducing specific functional groups or molecules into the side chains of hydrophilic natural polymers, reversible hydrogels can be constructed by forming non-covalent bonds to achieve the preparation of the pore plate gel material described in this invention.

[0079] This application presents the gel-sol transition of the hydrogel material by preparing the hydrogel material through non-covalent bonds.

[0080] In an embodiment of a porous plate, forming the non-covalent bonds includes achieving it through one or more of host-guest interactions, reversible hydrogen bonds, metal coordination, hydrophobic interactions, van der Waals forces, and π-π interactions.

[0081] Host-guest interaction refers to a complex composed of two or more molecules or ions in supramolecular chemistry, and these molecules or ions are combined together in a unique structural relationship through forces other than covalent bonds.

[0082] Host-guest interaction is an emerging technology, and through this interaction / technology, hydrogels that can undergo gel-sol transitions can be prepared.

[0083] Specifically, in the present invention, cyclodextrin and reduced ferrocene can be introduced into the hydrophilic natural polymer, respectively. The resulting hydrophilic natural polymer is then prepared into aqueous solutions. After the two solutions are evenly mixed, a host-guest interaction between the cyclodextrin and reduced ferrocene in the molecule can be used to produce a hydrogel material. The porous plate prepared from this hydrogel material can be used to culture cell aggregates. Subsequently, FeCl3 can be added to convert the reduced ferrocene into an oxidized ferrocene, achieving a gel-sol transition and converting the gel material into a sol. This mixture of cell aggregates and the natural polymer can then be collected by centrifugation to obtain uniformly sized cell aggregates.

[0084] Reversible hydrogen bonding refers to a covalent bond between a hydrogen atom and an atom X with high electronegativity. If a hydrogen atom comes into contact with an atom Y with high electronegativity and small radius (OFN, etc.), hydrogen is used as a medium between X and Y to form a special intermolecular or intramolecular interaction of the form XH…Y, which is called a hydrogen bond.

[0085] Specifically, in the present invention, poly(N-isopropylacrylamide) can be introduced into the hydrophilic natural polymer. After the resulting hydrophilic natural polymer is prepared into an aqueous solution, the solution can be heated to approximately 32°C to form strong intermolecular or intramolecular hydrogen bonds between the poly(N-isopropylacrylamide) chains to obtain a hydrogel material. The porous plate prepared with the hydrogel material can be used to culture cell aggregates. Subsequently, the gel-sol transition can be achieved by cooling the solution to convert the gel material into a sol, resulting in a mixed solution of cell aggregates and the natural polymer. Cell aggregates of uniform size can then be collected by centrifugation.

[0086] In an embodiment of a multi-well plate, a plurality of said well arrays are arranged on said plate, wherein Figures 2 to 6 The pores shown are arranged in an array of 4 rows and 6 columns; the diameter D of the pores is 200-1000 μm, the depth of the pores is 200-1000 μm, and the spacing S between two adjacent pores is 50-200 μm. It should be noted that the diameter of the pores described in this application is the equivalent circle diameter (area).

[0087] In an embodiment of a porous plate, the hydrophilic natural polymer is selected from one or a combination of two or more materials selected from hyaluronic acid, polyglutamic acid, chitosan, alginic acid and heparin; preferably, it is hyaluronic acid with a molecular weight of 40 kDa to 400 kDa.

[0088] In one embodiment of a multiwell plate, the cross-linking agent is cystamine dihydrochloride.

[0089] The cross-linking agent in this embodiment is cystamine dihydrochloride, a cross-linking agent containing a disulfide bond.

[0090] In an embodiment of the porous plate, the hydrophilic natural polymer reacts with an activator before crosslinking, and the activator activates the carboxyl groups in the hydrophilic natural polymer.

[0091] In an embodiment of the porous plate, the activator is EDC and NHS.

[0092] Activating the carboxyl groups in the hydrophilic natural polymer using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) can make the crosslinking process rapid and efficient.

[0093] This embodiment relates to a method for preparing a porous plate for culturing cell aggregates, including:

[0094] Dissolution step: Dissolve the hydrophilic natural polymer to prepare a polymer solution;

[0095] Crosslinking and molding step: Add a crosslinking agent to the polymer solution and cure it into a hydrogel on a mold, and the mold is provided with a plurality of columns, and the crosslinking agent contains one or more chemical bonds selected from Schiff base bonds, disulfide bonds, borate ester bonds and hydrazone bonds.

[0096] The manufacturing method of this embodiment can directly form the porous plate of the above embodiment on a mold provided with a plurality of columns, and the preparation process is convenient and efficient.

[0097] In an embodiment of the preparation method, the hydrophilic natural polymer is selected from a combination of one or more materials such as hyaluronic acid, polyglutamic acid, chitosan, alginic acid and heparin; preferably hyaluronic acid with a molecular weight of 40 kDa to 400 kDa.

[0098] In an embodiment of the preparation method, an activation step is further included before the crosslinking and molding step;

[0099] The activation step is to add an activator to the polymer solution;

[0100] The activator activates the carboxyl groups in the hydrophilic natural polymer.

[0101] In an embodiment of the preparation method, the activator is EDC and NHS.

[0102] Activating the carboxyl groups in the hydrophilic natural polymer using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) can make the crosslinking process rapid and efficient.

[0103] In an embodiment of the manufacturing method, the crosslinking agent is cystamine dihydrochloride.

[0104] In an embodiment of the preparation method, asFigure 4 , Figure 6 As shown in Figure 6 , the column 2-1 in the mold 2 is formed by lithographic silica molding or 3D printing molding; a plurality of the columns 2-1 are arranged in an array; the diameter D' of the column 2-1 is 200-1000 μm, the height of the column 2-1 is 200-1000 μm, and the spacing S' between two adjacent columns is 50-200 μm. It should be noted that the diameter of the column in this application is the (area) equivalent circular diameter.

[0105] The lithographic silica molding or 3D printing molding has high precision, and the pore sizes on the corresponding manufactured porous plate are uniform, which is beneficial to the preparation of cell aggregates with uniform sizes. At the same time, the lithographic silica molding or 3D printing molding is convenient for adjusting the size, which is beneficial to the preparation of cell aggregates with different sizes.

[0106] In an embodiment of the manufacturing method, it further includes a dialysis step of performing gradient dialysis on the gel using deionized water.

[0107] Through dialysis, the reagents that have not fully reacted during the crosslinking process can be removed, thereby preventing reagent residues from affecting the subsequent culture of cell aggregates.

[0108] This embodiment provides a preparation method for a porous plate for culturing cell aggregates, including:

[0109] Dissolve the hydrophilic natural polymer introduced with functional groups to prepare a polymer solution;

[0110] The polymer solution forms a hydrogel through the formation of dynamic covalent bonds between the introduced functional groups, thereby preparing the porous plate.

[0111] The specific preparation method can be similar to the above preparation method through a crosslinking agent. Add the polymer solution into the above-mentioned mold, and then under certain conditions, such as adjusting the pH and other conditions, form the corresponding polymer solution into the porous plate.

[0112] This embodiment provides a preparation method for a porous plate for culturing cell aggregates, including:

[0113] Dissolve the hydrophilic natural polymer introduced with functional groups to prepare a polymer solution;

[0114] The polymer solution forms a hydrogel through the formation of non-covalent bonds between the introduced functional groups, thereby preparing the porous plate.

[0115] The specific preparation method can be similar to the above preparation method through a crosslinking agent. Add the polymer solution into the above-mentioned mold, and then under certain conditions, such as adding an oxidant, irradiating with a specific wavelength, and other conditions, form the corresponding polymer solution into the porous plate.

[0116] This embodiment provides a method for using a porous plate for forming a hydrogel material by connecting polymer chains through dynamic covalent bonds, including:

[0117] A sterilization step of sterilizing the porous plate;

[0118] An inoculation and cultivation step of inoculating cells in the pores of the porous plate for cultivation;

[0119] A decomposition step of subjecting the porous plate to a gel-sol transition after forming cell aggregates to obtain a cell aggregate suspension;

[0120] A collection step of separating and collecting cell aggregates.

[0121] The porous plate in the above embodiment, after being sterilized and preparing cell aggregates, can obtain a mixed solution of cell aggregates and natural polymers through a gel-sol transition, thereby facilitating the separation and collection of cell aggregates.

[0122] In an embodiment of the usage method, the sterilization step is to soak the porous plate with alcohol and then replace it with PBS.

[0123] In an embodiment of the usage method, in the collection step, the cell aggregate suspension is centrifuged to separate the cell aggregates.

[0124] In an embodiment of the usage method, when the hydrogel material is formed by connecting the polymer chains of the hydrophilic natural polymer through disulfide bonds in dynamic covalent bonds, the decomposition step is to add a reducing agent containing a thiol group to the porous plate after forming cell aggregates; the reducing agent is preferably glutathione.

[0125] Specifically, the reaction mechanism between the plate body prepared with a cross-linking agent containing disulfide bonds and the reducing agent containing a thiol group is as follows:

[0126]

[0127] In an embodiment of the usage method, the concentration of the glutathione is 10 - 30 mmol / L, preferably 14 - 17 mmol / L.

[0128] The porous plate for culturing cell aggregates in the above embodiments, and its manufacturing and use methods. Regarding the porous plate capable of undergoing gel-sol transition, on the one hand, the porous plate can be prepared with high precision and convenience through a column array formed by lithographic silica molding or 3D printing; on the other hand, the hydrophilicity of natural polymers is utilized to promote spontaneous cell aggregation; on another hand, the porous plate can be rapidly decomposed under corresponding conditions to form a mixed solution of cell aggregates and natural polymers, and can be conveniently collected by centrifugation without damaging the morphology of the generated cell aggregates, thereby enabling the preparation of a large number of cell aggregates with uniform size at one time, which can be adapted to printing channels and has unique advantages in the application of three-dimensional tissue printing, especially in some microtissue constructs based on kenzan needle arrays that can only use cell aggregates with uniform size. Compared with the existing technical solutions, the size of the cultured and collected cell aggregates is more uniform and more rapid and efficient.

[0129] Example

[0130] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0131] Unless otherwise stated, the materials, reagents, etc. used in the following examples can all be obtained from commercial sources.

[0132] Example 1

[0133] Weigh 0.1 g of hyaluronic acid with a molecular weight of 43 kDa (a hydrophilic natural polymer, the amount of substance of the repeating unit is 2.64×10 -4 mol) powder and dissolve it in 1 mL of deionized water, stir and dissolve at room temperature. After the hyaluronic acid is completely dissolved, add EDC and NHS equivalent to the carboxyl groups in the hyaluronic acid molecule, and continue to react for 1 hour. Then add cystamine dihydrochloride (crosslinker) with half the amount of substance of the carboxyl groups in the hyaluronic acid molecule, that is, the molar ratio of the crosslinker to the repeating unit of hyaluronic acid is 0.5:1. After mixing evenly, pour the solution into a mold. The outer diameter of the column of the mold is 200 μm and the height is 250 μm. After reacting overnight, take out the cured gel from the mold and perform gradient dialysis with deionized water for 48 hours to obtain a porous plate for batch production of cell aggregates. The macroscopic size of the porous plate is 25000 μm×25000 μm×500 μm.

[0134] Soak the prepared porous plate in 75% alcohol for 24 hours for sterilization, and then replace it with PBS five times in a sterile environment and set aside. After digesting and collecting L929 cells, adjust the cell concentration to 10 5cells / mL, inoculated into the prepared sterilized multi-well plate. After 24 hours, the cells spontaneously aggregated to form aggregates with a diameter of about 110 microns. Add 500 μL of 10 mmol / L glutathione solution to the well plate. After 0.5 h, the multi-well plate was completely dissolved. The cell aggregate suspension was collected and centrifuged to obtain cell aggregates with uniform size. The obtained cell aggregates are as Figure 1 shown.

[0135] The differences between Examples 2-9 and Example 1 are only the weight, type and molecular weight of the (hydrophilic natural) polymer, the type of crosslinking agent, and the concentration of the glutathione solution. In addition, the amount of substance of the repeating unit of polyglutamic acid in Example 4 is 7.75×10 -4 , and the amount of substance of the repeating unit of sodium alginate in Example 4 is 5×10 -4 . See Table 1 for details.

[0136] Table 1: Usage of each reagent in the examples

[0137]

[0138] The experimental results of the above examples are as follows:

[0139] Uniform-sized cell aggregates were obtained in Examples 1-9. The situation of the cell aggregates in Example 1 is shown in detail in Figure 1 . [[ID=2(]]

[0140] In addition, the dissolution time of the multi-well plate after adding the reducing agent in Examples 1-9 is shown in Table 2:

[0141] Table 2: Dissolution time of the multi-well plate in the examples

[0142] Example Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Example 9 Time / h 0.5 0.45 0.7 0.5 0.5 1 1.5 0.2 0.2

[0143] As can be seen from the above table, the dissolution time of the multi-well plate in Examples 1-5 is 0.45-0.7 h, and the dissolution rate is relatively fast; the dissolution time in Examples 6-7 is 1-1.5 h, and the dissolution is slow, affecting the experimental efficiency; although the dissolution time is the fastest in Examples 8-9, cell death occurs in the outermost layer of the cell aggregates.

[0144] Comparative Example 1:

[0145] Weigh 0.1 g of dextran powder with a molecular weight of 50 kDa (the amount of substance of the repeating unit is 6.17×10 -4) Dissolve it in 1 mL of dimethyl sulfoxide, heat it to 50 °C and stir to dissolve. After the dextran is completely dissolved, add an equal amount of EDC and NHS to the number of repeating units of the dextran molecule. After stirring evenly, add dithiodipropionic acid equivalent to 0.5 times the number of repeating units of the dextran molecule. After mixing evenly, pour the solution into a mold and react overnight. Take out the cured gel from the mold and perform gradient dialysis with deionized water for 48 hours to obtain the well plate material.

[0146] In this comparative example, a culture method using the well plate prepared in this example for culturing cell aggregates is as follows: specifically, soak the well plate in 75% alcohol for 24 hours, and then use PBS to replace it five times in a sterile environment. After digesting and collecting L929 cells, adjust the cell concentration to 10 5 cells / mL with 1640 medium, inoculate it into the prepared well plate, and observe at 24 hours, 3 days and 7 days. The cells grow dispersedly and do not aggregate. According to this protocol, the dextran-based well plate material cannot achieve the preparation of cell aggregates.

[0147] In this comparative example, dextran is not a hydrophilic natural polymer and does not contain ionizable groups such as carboxyl groups, making its hydrophilicity relatively low. Although its solution can be used to prepare porous plates by crosslinking, it cannot inhibit protein adsorption, resulting in a strong interaction between cells and the material, and thus the cells cannot spontaneously aggregate.

[0148] Comparative Example 2:

[0149] Thaw the 3D-osteoblast aggregate reagent (3D-OSpS), fetal bovine serum and double antibody at 37 °C and then mix them to obtain the 3D-osteoblast aggregate medium (3D-OSpM). Collect the cells, resuspend them with 3D-OSpM and adjust the cell concentration to 3.2×10 5 cell / mL. Add 200 μL per well to a 96-well plate and culture it at 37 °C and 5% CO2. Replace the top 60 - 70% of the culture medium with fresh medium every 4 days. Observe under a microscope at 3 days, and the photo is as Figure 7 shown. When the generated cell aggregates need to be collected for use, use a pipette to aspirate the cell aggregates in each well into a 50 mL centrifuge tube in sequence, wash them with PBS, and then aspirate the remaining cell aggregates in each well into a 50 mL centrifuge tube in sequence. Observe whether there are remaining cell aggregates. If there are remaining, continue the above PBS washing operation and collect the remaining cell aggregates. Centrifuge and pour out the liquid to obtain the cell aggregates.

[0150] This comparative example gives a currently common method for preparing cell aggregates, but as Figure 7As shown, the cell aggregate sizes are non-uniform. Additionally, the collection process requires multiple uses of PBS to wash each well, which is time-consuming and cumbersome.

[0151] The above examples are only for illustrating the technical concept and features of the present invention. The purpose is to enable those who are familiar with this technology to understand the content of the present invention and implement it accordingly, and it should not be used to limit the protection scope of the present invention. All equivalent transformations or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A multi-well plate for culturing cell aggregates, comprising: A plate body, wherein the material forming the plate body is a hydrogel material prepared from a hydrophilic natural polymer, and the hydrogel material can undergo a gel-sol transition; a hole body, the hole body being provided on at least one surface of the plate body; The hydrogel material is formed by connecting the hydrophilic natural polymer chains through dynamic covalent bonds; The dynamic covalent bond includes one or more of a Schiff base bond, a disulfide bond, a borate bond and an acylhydrazone bond.

2. The porous plate according to claim 1, wherein The hydrogel material is formed by cross-linking the hydrophilic natural polymer using a cross-linking agent, wherein the cross-linking agent contains dynamic covalent bonds.

3. The porous plate according to claim 1, wherein Functional groups are introduced into the hydrophilic natural polymer, and the hydrophilic natural polymer forms the hydrogel material by forming dynamic covalent bonds between the introduced functional groups.

4. The porous plate according to claim 1, wherein A plurality of hole arrays are arranged on the plate; The diameter of the pores is 200 to 1000 μm, the depth of the pores is 200 to 1000 μm, and the distance between two adjacent pores is 50 to 200 μm.

5. The porous plate according to claim 1, wherein The hydrophilic natural polymer is selected from one or a combination of two or more materials selected from hyaluronic acid, polyglutamic acid, chitosan, alginic acid and heparin.

6. The porous plate according to claim 5, wherein The hydrophilic natural polymer is hyaluronic acid with a molecular weight of 40 kDa to 400 kDa.

7. The porous plate according to claim 2, wherein The cross-linking agent is cystamine dihydrochloride.

8. The porous plate according to claim 2, wherein The hydrophilic natural polymer reacts with an activator before cross-linking, and the activator activates the carboxyl groups in the hydrophilic natural polymer.

9. The porous plate according to claim 8, wherein The activating agents are 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

10. A method for preparing a multi-well plate for culturing cell aggregates, comprising: Dissolution step: dissolving the hydrophilic natural polymer to prepare a polymer solution; Cross-linking molding step: adding a cross-linking agent to the polymer solution and solidifying and molding it into a hydrogel on a mold to prepare the porous plate, wherein a plurality of columns are provided in the mold, and the cross-linking agent contains one or more chemical bonds selected from the group consisting of Schiff base bonds, disulfide bonds, borate bonds, and acylhydrazone bonds.

11. The preparation method according to claim 10, characterized in that The hydrophilic natural polymer is selected from one or a combination of two or more materials selected from hyaluronic acid, polyglutamic acid, chitosan, alginic acid and heparin.

12. The preparation method according to claim 11, characterized in that The hydrophilic natural polymer is hyaluronic acid with a molecular weight of 40 kDa to 400 kDa.

13. The preparation method according to claim 10, wherein The cross-linking molding step also includes an activation step before the cross-linking molding step; The activation step is to add an activator to the polymer solution; The activator activates the carboxyl groups in the hydrophilic natural polymer.

14. The preparation method according to claim 13, wherein The activating agents are 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

15. The preparation method according to claim 10, wherein The cross-linking agent is cystamine dihydrochloride.

16. The preparation method according to claim 10, wherein The column in the mold is formed by photolithography silicon dioxide molding or 3D printing; The plurality of columns are arranged in an array; The diameter of the column is 200-1000 μm, the height of the column is 200-1000 μm, and the distance between two adjacent columns is 50-200 μm.

17. The preparation method according to claim 10, wherein The method further includes a dialysis step in which the hydrogel is subjected to gradient dialysis using deionized water.

18. A method for preparing a multi-well plate for culturing cell aggregates, comprising: Dissolution step: dissolving the hydrophilic natural polymer with introduced functional groups to prepare a polymer solution; Molding step: in a mold, the polymer solution forms a hydrogel by forming dynamic covalent bonds between the introduced functional groups, thereby preparing the porous plate; The dynamic covalent bond includes one or more of a Schiff base bond, a disulfide bond, a borate bond and an acylhydrazone bond.

19. The method for using the multi-well plate according to claim 1, comprising: a sterilization step of sterilizing the multi-well plate; an inoculation and culturing step, inoculating cells into the wells of the multi-well plate and culturing them; a decomposition step, after forming cell aggregates, converting the multi-well plate into a gel-sol to obtain a cell aggregate suspension; In the collection step, cell aggregates are separated and collected.

20. The method of use according to claim 19, wherein: The sterilization step is to soak the multi-well plate with alcohol and then replace it with PBS.

21. The method of use according to claim 19, wherein: In the collecting step, the cell aggregate suspension is centrifuged to separate the cell aggregates.

22. The method of use according to claim 19, wherein: When the hydrogel material is formed by connecting the hydrophilic natural polymer chains via disulfide bonds in dynamic covalent bonds, the decomposition step is to add a reducing agent containing a thiol group to the multi-well plate after the cell aggregates are formed.

23. The method of use according to claim 22, wherein: The reducing agent is glutathione.

24. The method of use according to claim 23, wherein: The concentration of the glutathione is 10 to 30 mmol / L.

25. The method of use according to claim 24, wherein: The concentration of the glutathione is 13-17 mmol / L.

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