Hydrogel microarray preparation device and method and organoid cell mass culture method

Abstract

The invention discloses a hydrogel microarray preparation device and method and an organoid cell mass culture method, and relates to the technical field of organoid culture. The invention aims to solve the problems of low culture efficiency, poor consistency and low automatic compatibility in the existing process of culturing the organoid in an array manner. The microarray device comprises a bottom die, a plurality of upper dies and a plurality of array retainers, the upper end face of the bottom die is horizontally arranged, the array retainers are installed on the upper end face of the bottom die, one upper die is arranged over each array retainer, downward protrusions in an array shape are evenly distributed in the middle of the lower end face of each upper die, and the lower end face of each upper die is provided with a plurality of lower protrusions. And the downward bulge is arranged towards the middle part of the upper end of the array retainer. The method is used for organoid culture.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of organoid culture, and particularly relates to a hydrogel microarray preparation device suitable for automated culture, a hydrogel microarray preparation method and an organoid cell cluster culture method. BACKGROUND

[0002] As a three-dimensional cell model in vitro, organoids have important value in disease research, drug screening and regenerative medicine. Traditional organoid culture relies on manual operation (such as the Matrigel droplet method), which has the following problems: 1. Batch difficulty: low culture efficiency, difficult to meet the high-throughput demand; 2. Poor consistency: manual operation leads to significant differences in microenvironment parameters; 3. Low automation compatibility: traditional methods require frequent transfer of organoids, which easily loses spatial information and is difficult to integrate with automated equipment. SUMMARY

[0003] In order to solve the problems of low culture efficiency, poor consistency and low automation compatibility in the process of existing array culture organoids, the present application further provides a hydrogel microarray preparation device suitable for automated culture, a hydrogel microarray preparation method and an organoid cell cluster culture method.

[0004] The technical solution adopted by the present application to solve the above technical problems is as follows:

[0005] The first aspect of the embodiment of the present application provides a hydrogel microarray preparation device, which comprises a bottom mold, a plurality of upper molds and a plurality of array holding frames. The upper end surface of the bottom mold is horizontally arranged. The plurality of array holding frames are respectively installed on the upper end surface of the bottom mold. An upper mold is arranged above each array holding frame. The lower end surface of the upper mold is uniformly provided with downward protrusions in an array shape. The downward protrusions are arranged towards the middle part of the upper end of the array holding frame.

[0006] In a possible design, the shape of the upper end surface of the downward protrusion is a polygon, and the bottom of the downward protrusion is a sharp end.

[0007] In a possible design, the array holding frame comprises an array ring and two clamping ear plates. The array ring is arranged on the upper end surface of the bottom mold, and the lower end surface of the array ring is flush with the upper end surface of the bottom mold. The two clamping ear plates are respectively and oppositely fixed to the two sides of the outside of the array ring, and the two clamping ear plates are clamped on the two sides of the upper end surface of the bottom mold. The lower end surface of the upper mold is arranged in the array ring.

[0008] In a possible design, the shape of the clamping ear plate is L-shaped. The horizontal end of the clamping ear plate is fixed to the array ring, and the inner side surface of the vertical end of the clamping ear plate is provided with a clamping protrusion.

[0009] In a possible design, a plurality of anti-falling plates are fixed to the middle part of the inner side wall of the array ring in the circumferential direction.

[0010] In a possible design, the four peripheral edges of the lower end surface of the upper mold are uniformly provided with a plurality of limiting blocks in the circumferential direction, and each limiting block is arranged between two adjacent anti-disengagement plates, and the outer side surface of the limiting block is matched with the inner side surface of the arrayed annular ring.

[0011] In a possible design, the middle part of the lower end surface of the upper mold is provided with an arrayed boss, and the arrayed downward protrusions are arranged on the lower end surface of the arrayed boss.

[0012] In a possible design, the lower end surface of the upper mold is provided with a marking protrusion, and the marking protrusion is arranged on one side of the arrayed downward protrusions.

[0013] The second aspect of the embodiment of the present application provides a hydrogel microarray preparation method based on the hydrogel microarray preparation device provided in the first aspect, and the method comprises the following steps:

[0014] The bottom mold is placed on the platform, and then a plurality of array holding frames are clamped on the corresponding positions on the upper end surface of the bottom mold one by one;

[0015] The hydrogel material is heated to be in a fluid state, the hydrogel material in the fluid state is added into the arrayed annular ring of the array holding frame, and then the upper mold is pressed in the arrayed annular ring of the array holding frame to complete the injection of the hydrogel material in the array holding frame;

[0016] The injection of the hydrogel material is completed in the plurality of array holding frames one by one;

[0017] After the hydrogel material in the array holding frame is completely solidified, the upper mold is removed, wherein the upper surface of the solidified hydrogel material forms an arrayed pit under the action of the arrayed downward protrusions, so as to obtain the hydrogel microarray on the array holding frame.

[0018] The third aspect of the embodiment of the present application provides a kind of organoid cell cluster culture method based on hydrogel microarray, comprising:

[0019] The array holding frame with hydrogel microarray is clamped by mechanical hand and placed in automatic culture instrument, and source cell suspension is added into the pit of hydrogel microarray by pipetting mechanism, so that source cell suspension is settled to the bottom of pit under the action of gravity, and is gathered to the center at the bottom, and source cell suspension in hydrogel microarray is aggregated into cluster under culture condition, and forms cell cluster.

[0020] The array holding frame with hydrogel microarray is transferred from automatic culture instrument to dehydration module by mechanical hand for dehydration;

[0021] After dehydration, the Matrigel stored at 0℃ is added into the recesses of the hydrogel microarray to wrap the cell clusters at the bottom of the recesses, and a culture dish is inverted on the Matrigel so that the bottom of the culture dish is in contact with the Matrigel, and then the mechanical hand is used to pick up the constant-temperature heat block and stick to the outer bottom of the culture dish to keep the constant-temperature heat block at 37℃, thereby assisting the Matrigel to solidify;

[0022] After solidification, the culture dish is clamped by the mechanical hand, and the solidified Matrigel is separated from the hydrogel microarray;

[0023] The cell clusters wrapped by the solidified Matrigel in the culture dish retain the original spatial information, so that subsequent organoid culture can be continued in the culture dish.

[0024] Compared with the prior art, the application has the beneficial effects that:

[0025] The application provides a hydrogel microarray preparation device, method and organoid cell cluster culture method suitable for automatic culture, which can realize batch array culture of organoids in a single culture dish or well plate, greatly reduces the operation times of experimenters, improves the efficiency and the consistency of the culture process. Meanwhile, the technology can be adapted to an automatic culture system, so that the automatic and batch culture of organoids with good consistency in growth state can be realized. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a structural schematic diagram of a hydrogel microarray preparation device suitable for automatic culture provided by an embodiment of the application;

[0027] Figure 2 is a structural schematic diagram of an upper mold involved in the embodiment of the application;

[0028] Figure 3 is a structural schematic diagram of an array holder involved in the embodiment of the application;

[0029] Figure 4 is a structural schematic diagram of an array holder with a hydrogel microarray involved in the embodiment of the application;

[0030] Figure 5 is a structural schematic diagram of the mechanical hand clamping the two sides of the array ring involved in the embodiment of the application;

[0031] Figure 6 is a structural schematic diagram of the mechanical hand clamping the two sides of the clamping ear plate involved in the embodiment of the application.

[0032] In the attached diagram, 1-upper mold, 11-downward protrusion, 12-limiting block, 13-marking protrusion, 14-array protrusion; 2-array retainer, 21-array ring, 22-clamping ear plate, 23-anti-detachment plate, 24-clamping protrusion; 3-hydrogel microarray, 31-recess; 4-bottom mold. Detailed Implementation

[0033] To make the technical problems solved, technical solutions, and beneficial effects of this application clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining this application and are not intended to limit this application.

[0034] Current array-based organoid culture techniques commonly involve adding treated source cells into multi-well plates with a U-shaped bottom (such as 96-well or 384-well plates). Once the cells clump together, they are transferred from the wells using a pipette, mixed with Matrigel, and then seeded into the culture plate. This culture method has several problems:

[0035] Low cultivation efficiency: Manual single-well operation is slow, inefficient, and difficult to scale up, making it difficult to meet high-throughput requirements;

[0036] Poor consistency: There are many uncertainties in manual operation, resulting in significant differences;

[0037] Low automation compatibility: Transferred cell clusters are prone to losing spatial information, making it difficult to observe and track them in real time from the source cell line, and it is also difficult to integrate with automated equipment.

[0038] In view of this, embodiments of this application provide a hydrogel microarray preparation device adapted for automated culture, a method for preparing a hydrogel microarray based on the device, and a method for culturing organoid cell clusters based on the hydrogel microarray, in order to solve the above-mentioned technical problems. The hydrogel preparation device adapted for automated culture provided in embodiments of this application includes:

[0039] The bottom mold 4, multiple upper molds 1 and multiple array retainers 2 are provided. The upper end face of the bottom mold 4 is horizontally arranged. Multiple array retainers 2 are respectively installed on the upper end face of the bottom mold 4. Each array retainer 2 is provided with an upper mold 1 directly above it. The lower end face of the upper mold 1 is evenly distributed with downward protrusions 11 in an array. The downward protrusions 11 are arranged towards the middle of the upper end of the array retainer 2.

[0040] In the embodiment of this application, multiple array holders 2 can be clamped on the bottom mold 4, allowing multiple hydrogel microarrays to be fabricated at once. Through the cooperation of the bottom mold 4 and the upper mold 1, the hydrogel microarray 3 is fabricated within the array holder 2. The lower end face of the upper mold 1 is evenly provided with an array of downward protrusions 11, forming an array of pits 31 on the upper surface of the hydrogel microarray 3, which facilitates the batch array culture of organoids.

[0041] In some embodiments, the shape of the upper end surface of the downward protrusion 11 is polygonal, and the bottom of the downward protrusion 11 is pointed. The downward protrusion 11 can allow the cells entering the pit 31 to naturally settle under the action of gravity. The pointed bottom of the downward protrusion 11 can facilitate the water gel material to quickly gather at the bottom under the action of gravity. The common shape of the micro-pore for culturing cell aggregates is generally circular from the top view, and the circular pore array is arranged on a plane, and there will be a gap plane between the pores. The cells in the cell suspension cannot gather into the pores when falling on the plane. In this embodiment, the shape of the pit 31 formed by the downward protrusion 11 on the water gel microarray 3 is polygonal from the top view, that is, the pits 31 are closely arranged on the planting plane according to the same number of pores closest to the single pore in the array, so that the cells in the cell suspension can completely fall into the pits 31, which not only improves the utilization rate of cells, but also makes the calculation of the number of cells in a single pore more accurate.

[0042] In some embodiments, the array holder 2 includes an array ring 21 and two clamping lug plates 22. The array ring 21 is arranged on the upper end surface of the bottom mold 4, and the lower end surface of the array ring 21 is flush with the upper end surface of the bottom mold 4. The two clamping lug plates 22 are respectively and oppositely fixed to the two sides outside the array ring 21, and are clamped on the two sides of the upper end surface of the bottom mold 4. The lower end surface of the upper mold 1 is arranged in the array ring 21. Under the action of the upper mold 1 and the bottom mold 4, after the water gel material is injected into the array ring 21, the lower end surface of the water gel microarray 3 formed is smooth and horizontal, and the upper end surface has a matrix-shaped closely arranged pit 31. When taking and placing are needed in manual or automated culture operation, the two clamping lug plates 22 can be clamped on the two sides of the array ring 21 for operation, and the two clamping lug plates 22 can also be clamped on the two sides of the clamping lug plate 22 for operation in a 6-well plate or a φ35 culture dish.

[0043] In some embodiments, the clamping lug plate 22 is L-shaped, the horizontal end of the clamping lug plate 22 is fixed to the array ring 21, and the inner side surface of the vertical end of the clamping lug plate 22 is provided with a clamping protrusion 24 to realize the clamping between the array holder 2 and the bottom mold 4.

[0044] In some embodiments, a plurality of anti-falling plates 23 are uniformly fixed to the middle part of the inner side wall of the array ring 21 in the circumferential direction. The anti-falling plates 23 can prevent the water gel from falling out after solidification after the water gel material is injected.

[0045] In some embodiments, a plurality of limiting blocks 12 are uniformly arranged on the peripheral edge of the lower end surface of the upper mold 1 in the circumferential direction, each limiting block 12 is arranged between two adjacent anti-falling plates 23, and the outer side surface of the limiting block 12 cooperates with the inner side surface of the array ring 21. The limiting block 12 can assist the upper mold 1 to accurately press on the array holder 2.

[0046] In some embodiments, an array of protrusions 14 is provided in the middle of the lower end face of the upper mold 1, and an array of downward protrusions 11 are all provided on the lower end face of the array protrusions 14. After pressing, the array protrusions 14 can form a concave planting plane on the upper surface of the hydrogel microarray 3, and the pits 31 are all within the planting plane. When adding source cell suspension and matrix gel, the overall volume of the concave area can be obtained through theoretical calculation, which facilitates the quantitative addition of cell suspension and matrix gel during the culture operation.

[0047] In some embodiments, a marking protrusion 13 is provided on the lower end face of the upper mold 1, and the marking protrusion 13 is disposed on one side of the array of downward protrusions 11. After pressing, the marking protrusion 13 can form a mark on the upper surface of the hydrogel microarray 3, which is used to analyze and record the spatial position information of each pore.

[0048] In accordance with the above-mentioned apparatus for preparing hydrogel microarrays adapted for automated culture, this application also provides a method for preparing hydrogel microarrays adapted for automated culture, comprising:

[0049] Place the bottom mold 4 on the platform, and then snap multiple array holders 2 one by one into the corresponding positions on the upper surface of the bottom mold 4;

[0050] The hydrogel material is heated to make it fluid, and the fluid hydrogel material is added into the array ring 21 of the array holder 2. Then the upper mold 1 is pressed into the array ring 21 of the array holder 2 to complete the injection of the hydrogel material into the array holder 2.

[0051] Hydrogel material was injected sequentially into each of the multiple array holders 2.

[0052] After the hydrogel material inside the array holder 2 has completely solidified, the upper mold 1 is removed. The upper surface of the solidified hydrogel material forms an array of pits 31 under the action of the array of downward protrusions 11, so as to obtain a hydrogel microarray 3 on the array holder 2.

[0053] The preparation method provided in this application embodiment can obtain an array holder 2 with hydrogel microarray 3, and transfer the array holder 2 and the hydrogel microarray 3 thereon to realize the subsequent batch array culture of organoids in a single culture dish or well plate, which greatly reduces the number of operations by the experimenter, improves efficiency, and improves the consistency of the culture process.

[0054] Based on the hydrogel microarray prepared by the above scheme, this application embodiment also provides a method for culturing organoid cell clusters based on hydrogel microarrays, including:

[0055] The array holder 2 with hydrogel microarray 3 is picked up by a robotic arm and placed in an automated culture instrument. The source cell suspension is added into the pit 31 of the hydrogel microarray 3 by a pipetting mechanism, so that the source cell suspension settles to the bottom of the pit 31 under the action of gravity and gathers from the bottom to the center. The source cell suspension in the hydrogel microarray 3 aggregates into clusters under culture conditions to form cell clusters.

[0056] A robotic arm is used to transfer the array holder 2 with the hydrogel microarray 3 from the automated culture instrument to the dehydration module for dehydration;

[0057] After dehydration, a matrix gel stored at 0℃ was added to the pit 31 of the hydrogel microarray 3, so that the matrix gel could wrap the cell clusters at the bottom of the pit 31. A culture dish was then placed upside down on the matrix gel so that the bottom of the culture dish was in contact with the matrix gel. Then, a constant temperature heat conduction block was picked up by a robotic arm and attached to the bottom of the culture dish. The constant temperature heat conduction block was kept at a constant temperature of 37℃ to assist the matrix gel in curing.

[0058] After curing, the culture dish is picked up by a robotic arm, and the matrix adhesive is cured at the bottom of the culture dish. The cured matrix adhesive is separated from the hydrogel microarray 3.

[0059] The cell clusters encapsulated by the matrix gel solidified in the culture dish retain their original spatial information, allowing for continued organoid culture within the dish.

[0060] In this embodiment, hydrogel microarrays are used to culture organoid cell clusters, reducing the number of operator interventions, improving efficiency, and enhancing the consistency of the culture process. Furthermore, it can be adapted to automated culture systems, thereby enabling automated, batch culture of organoids with consistent growth states.

[0061] To further illustrate the embodiments of this application, the following description is provided in conjunction with the accompanying drawings. Figures 1 to 6 A detailed description is provided below. It is understood that the same or corresponding content in the above embodiments and the following specific embodiments can be referenced interchangeably, and will not be repeated hereafter.

[0062] Specific implementation method one: Combining Figures 1 to 6 This embodiment describes a hydrogel microarray preparation device adapted for automated culture, comprising a bottom mold 4, multiple upper molds 1, and multiple array holders 2. The upper surface of the bottom mold 4 is horizontally arranged, and the multiple array holders 2 are respectively installed on the upper surface of the bottom mold 4. Each array holder 2 is provided with an upper mold 1 directly above it. The lower surface of the upper mold 1 is evenly distributed with an array of downward protrusions 11 in the middle, and the downward protrusions 11 are arranged towards the middle of the upper end of the array holder 2.

[0063] The plurality of array holders 2 are arranged along the length direction of the bottom mold 4 and clamped on the upper end surface of the bottom mold 4.

[0064] The upper mold 1 is an upper mold made by 3D printing, CNC, etc., and is used for imprinting a microarray structure.

[0065] The bottom mold 4 serves as a limiting structure, which can fix the position of the array holder 2 and make the bottom surface of the hydrogel microarray 3 horizontal.

[0066] In the embodiment, a plurality of array holders 2 can be clamped on the bottom mold 4, and a plurality of hydrogel microarrays can be completed at one time. Through the cooperation of the bottom mold 4 and the upper mold 1, the hydrogel microarray 3 is made in the array holder 2. The lower end surface of the upper mold 1 is uniformly provided with downward protrusions 11 in an array, and the upper surface of the hydrogel microarray 3 is formed with array-shaped pits 31, so as to realize batch array culture of organoids.

[0067] Specific implementation method two: combined with Figure 2 In the embodiment, the upper end surface of the downward protrusion 11 is polygonal, and the bottom of the downward protrusion 11 is a sharp end.

[0068] The technical features not disclosed in the embodiment are the same as those in the specific implementation method one.

[0069] In the embodiment, the downward protrusions 11 in an array are closely arranged.

[0070] The downward protrusion 11 can make the cells entering the pit 31 naturally settle under the action of gravity.

[0071] In the embodiment, the shape of the downward protrusion 11 is an inverted pyramid. For example, an inverted triangular pyramid, an inverted quadrangular pyramid, and an inverted hexagonal pyramid, the upper end surface shapes of which are a regular triangle, a square, and a regular hexagon respectively, and the cross-sectional shape of the bottom is U-shaped or V-shaped.

[0072] The shape of the common micro-pore for culturing cell clusters is generally circular from the perspective of top view. The circular pore array is arranged on a plane, and there will be a gap plane between the pores. The cells in the cell suspension cannot gather into the pores when falling on the plane. In the embodiment, the shape of the pit 31 formed by the downward protrusion 11 on the hydrogel microarray 3 is polygonal from the perspective of top view, according to the number of the same pores closest to the single pore in the array. That is, the pits 31 are closely arranged on the planting plane, so that the cells in the cell suspension can completely fall into the pits 31. Both the utilization rate of cells and the calculation accuracy of the number of cells in a single pore are improved.

[0073] Specific implementation method three: combined with Figure 3 、 Figure 5 and Figure 6In this embodiment, the array holder 2 comprises an array ring 21 and two clamping lugs 22. The array ring 21 is arranged on the upper end surface of the bottom mold 4, and the lower end surface of the array ring 21 is flush with the upper end surface of the bottom mold 4. The two clamping lugs 22 are respectively fixed to the two sides of the outer part of the array ring 21, and are clamped on the two sides of the upper end surface of the bottom mold 4. The lower end surface of the upper mold 1 is arranged in the array ring 21.

[0074] The technical features not disclosed in this embodiment are the same as those in the first embodiment.

[0075] The shape of the lower end of the upper mold 1 is cylindrical, so that the shape of the lower end surface matches that of the array ring 21.

[0076] Under the action of the upper mold 1 and the bottom mold 4, after the array ring 21 is injected with the hydrogel material, the lower end surface of the hydrogel microarray 3 formed thereby is smooth and horizontal, and the upper end surface has a matrix of closely arranged pits 31.

[0077] When the array holder 2 needs to be taken out or placed in during manual or automated culture operation, it can be clamped from the two sides of the array ring 21, or from the two sides of the clamping lugs 22.

[0078] Embodiment Four: Combination Figures 3 to 4 In this embodiment, the clamping lug 22 is L-shaped, the horizontal end of the clamping lug 22 is fixed to the array ring 21, and the inner side surface of the vertical end of the clamping lug 22 is provided with a clamping protrusion 24.

[0079] The technical features not disclosed in this embodiment are the same as those in the third embodiment.

[0080] This design achieves clamping between the array holder 2 and the bottom mold 4.

[0081] Embodiment Five: Combination Figure 3 In this embodiment, the inner side wall of the array ring 21 is provided with a plurality of anti-falling plates 23 evenly distributed in the circumferential direction at the middle part.

[0082] The technical features not disclosed in this embodiment are the same as those in the third embodiment.

[0083] The anti-falling plates 23 are arranged at the middle part of the inner side wall of the array ring 21, which can prevent the hydrogel from falling out after solidification after being injected with the hydrogel material.

[0084] Embodiment Six: Combination Figure 2In this embodiment, the lower end surface of the upper mold 1 is provided with a plurality of limiting blocks 12 distributed along the circumferential direction, and each limiting block 12 is arranged between two adjacent anti-disengagement plates 23.

[0085] The technical features not disclosed in this embodiment are the same as those in Embodiment Five.

[0086] The limiting blocks 12 can assist the upper mold 1 to be accurately pressed on the array holder 2.

[0087] Embodiment Seven: Combination Figure 2 In this embodiment, the middle part of the lower end surface of the upper mold 1 is provided with an array boss 14, and the array-shaped downward protrusions 11 are arranged on the lower end surface of the array boss 14.

[0088] The technical features not disclosed in this embodiment are the same as those in Embodiment One.

[0089] After the array boss 14 is pressed, a concave planting plane can be formed on the upper surface of the hydrogel microarray 3, and the pits 31 are in the planting plane. When the source cell suspension and the matrix glue are added subsequently, the overall volume of the concave part can be calculated, which facilitates the quantitative addition of cell suspension and matrix glue in the cultivation operation.

[0090] Embodiment Eight: Combination Figure 2 In this embodiment, the lower end surface of the upper mold 1 is provided with a marking protrusion 13, and the marking protrusion 13 is arranged on one side of the array-shaped downward protrusions 11.

[0091] The technical features not disclosed in this embodiment are the same as those in Embodiment One.

[0092] After the marking protrusion 13 is pressed, a mark can be formed on the upper surface of the hydrogel microarray 3, which is used for analyzing and recording the spatial position information of each hole.

[0093] Embodiment Nine: Combination Figures 1 to 6 In this embodiment, a hydrogel microarray preparation method suitable for automated culture includes:

[0094] Step One, mold positioning: place the bottom mold 4 on the platform, and then sequentially clamp the plurality of array holders 2 on the corresponding positions on the upper end surface of the bottom mold 4;

[0095] Step Two, inject hydrogel: heat the hydrogel material to make it in a fluid state, add the hydrogel material in a fluid state into the array circular ring 21 of the array holder 2, and then press the upper mold 1 on the array circular ring 21 of the array holder 2 to complete the injection of the hydrogel material in the array holder 2.

[0096] Step three, injection of hydrogel material is completed one by one in the multiple array holders 2 in sequence respectively;

[0097] Step four, solidification molding: after the hydrogel material in the array holder 2 is completely solidified, the upper mold 1 is removed, wherein the upper surface of the solidified hydrogel material forms an array of pits 31 under the action of the array-like downward protrusion 11, so as to obtain a hydrogel microarray 3 on the array holder 2.

[0098] The hydrogel in the embodiment comprises one of the following three materials:

[0099] i. a component of natural origin selected from the group comprising polysaccharides, gelatinous proteins, agarose, alginate, chitosan, dextran, gelatin, laminin, collagen, hyaluronic acid, fibrin or mixtures thereof, or from the group of matrices derived from complex tissues consisting of Matrigel, basement membrane and Cartigel, preferably wherein in the gel the concentration of collagen is between 0.4 mg / ml and 3.6 mg / ml and Matrigel is at a percentage between 10% (v / v) and 90% (v / v);

[0100] ii. a cross-linked synthetic hydrophilic polymer functionalized with proteins or peptides derived from the extracellular matrix (ECM), preferably wherein the hydrophilic polymer is selected from the group comprising poly(ethylene glycol), poly(oxazoline), poly(aliphatic urethane), poly(ether urethane), poly(ester urethane), poly(ethylene copolymer), polyamide, polyvinyl alcohol, poly(ethylene oxide), polypropylene oxide, polypropylene glycol, polytetrahydrofuran, polyvinylpyrrolidone, polyacrylamide, poly(hydroxyethyl methacrylate), poly(hydroxyethyl methacrylate) and mixtures or copolymers thereof;

[0101] iii. a combination of natural delivery and synthetic precursors.

[0102] DETAILED DESCRIPTION TEN: COMBINATION Figures 1 to 6 In this embodiment, the hydrogel microarray-based organoid cell cluster culture method comprises:

[0103] First step, cell ball aggregation: the array holder 2 with the hydrogel microarray 3 is clamped by a mechanical hand and placed in an automated culture instrument, and a source cell suspension is added to the pit 31 of the hydrogel microarray 3 by a pipetting mechanism, so that the source cell suspension is settled to the bottom of the pit 31 under the action of gravity and gathered to the center at the bottom, and the source cell suspension in the hydrogel microarray 3 is aggregated into a cluster under the culture condition to form a cell cluster;

[0104] Second step, dehydration: the array holder 2 with the hydrogel microarray 3 is transferred from the automated culture instrument to the dehydration module by the mechanical hand for dehydration;

[0105] Third step, Matrigel solidification: after the completion of dehydration, Matrigel stored at 0℃ is added into the pits 31 of the hydrogel microarray 3 to wrap the cell clusters at the bottom of the pits 31, and a culture dish is inverted on the Matrigel to make the bottom of the culture dish contact with the Matrigel, then a constant temperature heat conducting block is picked up by the mechanical hand and attached to the outer bottom of the culture dish to keep the constant temperature heat conducting block at 37℃ to assist the solidification of the Matrigel;

[0106] Fourth step, separation of hydrogel: after the completion of solidification, the culture dish is gripped by the mechanical hand, the solidified Matrigel is at the bottom of the culture dish, and the solidified Matrigel is separated from the hydrogel microarray 3;

[0107] Fifth step, subsequent culture: the cell clusters wrapped by the solidified Matrigel in the culture dish retain the original spatial information, so that subsequent organoid culture is continued in the culture dish.

[0108] The above shows and describes the basic principles and main features of the present application and the advantages of the present application. It should be understood by those skilled in the art that the present application is not limited to the above embodiments, the above embodiments and descriptions in the specification are only to illustrate the principles of the present application, and various changes and improvements can be made to the present application without departing from the spirit and scope of the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A hydrogel microarray preparation device, characterized by, The utility model relates to a kind of array holding frame and array holding frame is used to the injection of hydrogel material. The upper end surface of the downward protrusion (11) is polygonal in shape, and the bottom of the downward protrusion (11) is a sharp end.

2. The hydrogel microarray preparation device of claim 1, wherein, The array holding frame (2) includes an array ring (21) and two clamping ear plates (22). The array ring (21) is arranged on the upper end surface of the bottom mold (4), and the lower end surface of the array ring (21) is flush with the upper end surface of the bottom mold (4). The two clamping ear plates (22) are respectively fixed to the two sides of the outer part of the array ring (21), and are clamped on the two sides of the upper end surface of the bottom mold (4). The lower end surface of the upper mold (1) is arranged in the array ring (21).

3. The hydrogel microarray preparation device of claim 1, wherein, The clamping ear plate (22) is L-shaped, and the horizontal end of the clamping ear plate (22) is fixed to the array ring (21). A clamping protrusion (24) is arranged on the inner side surface of the vertical end of the clamping ear plate (22).

4. The hydrogel microarray preparation device of claim 3, wherein, A plurality of anti-disengagement plates (23) are fixed to the middle part of the inner side wall of the array ring (21) in the circumferential direction.

5. The hydrogel microarray preparation device of claim 3, wherein, A plurality of limiting blocks (12) are arranged on the periphery of the lower end surface of the upper mold (1) in the circumferential direction. Each limiting block (12) is arranged between two adjacent anti-disengagement plates (23). The outer side surface of the limiting block (12) is matched with the inner side surface of the array ring (21).

6. The hydrogel microarray preparation device of claim 5, wherein, An array boss (14) is arranged on the middle part of the lower end surface of the upper mold (1). The array-shaped downward protrusions (11) are arranged on the lower end surface of the array boss (14).

7. The hydrogel microarray preparation device of claim 1, wherein, A marking protrusion (13) is arranged on the lower end surface of the upper mold (1). The marking protrusion (13) is arranged on one side of the array-shaped downward protrusions (11).

8. The hydrogel microarray preparation device of claim 1, wherein, The utility model relates to a kind of array holding frame and array holding frame is used to the injection of hydrogel material.

9. A method for preparing a hydrogel microarray of the hydrogel microarray preparation device according to any one of claims 1 to 8, characterized by, The array holding frame (2) includes an array ring (21) and two clamping ear plates (22). The array ring (21) is arranged on the upper end surface of the bottom mold (4), and the lower end surface of the array ring (21) is flush with the upper end surface of the bottom mold (4). The two clamping ear plates (22) are respectively fixed to the two sides of the outer part of the array ring (21), and are clamped on the two sides of the upper end surface of the bottom mold (4). The lower end surface of the upper mold (1) is arranged in the array ring (21). The clamping ear plate (22) is L-shaped, and the horizontal end of the clamping ear plate (22) is fixed to the array ring (21). A clamping protrusion (24) is arranged on the inner side surface of the vertical end of the clamping ear plate (22). A plurality of anti-disengagement plates (23) are fixed to the middle part of the inner side wall of the array ring (21) in the circumferential direction. A plurality of limiting blocks (12) are arranged on the periphery of the lower end surface of the upper mold (1) in the circumferential direction. Each limiting block (12) is arranged between two adjacent anti-disengagement plates (23). The outer side surface of the limiting block (12) is matched with the inner side surface of the array ring (21). An array boss (14) is arranged on the middle part of the lower end surface of the upper mold (1). The array-shaped downward protrusions (11) are arranged on the lower end surface of the array boss (14).

10. A hydrogel microarray-based organoid cell cluster culture method, characterized by, A marking protrusion (13) is arranged on the lower end surface of the upper mold (1). The marking protrusion (13) is arranged on one side of the array-shaped downward protrusions (11). The utility model relates to a kind of array holding frame and array holding frame is used to the injection of hydrogel material. The array holder (2) with the hydrogel microarray (3) is gripped by a mechanical hand and placed in an automated culture instrument, and a source cell suspension is added into the pits (31) of the hydrogel microarray (3) by a pipetting mechanism, so that the source cell suspension is settled to the bottom of the pits (31) under the action of gravity and gathered to the center at the bottom, and the source cell suspension in the hydrogel microarray (3) is aggregated into clusters under a culture condition, to form cell clusters; The array holder (2) with the hydrogel microarray (3) is transferred from the automated culture instrument to a dehydration module by a mechanical hand for dehydration; After the dehydration is completed, Matrigel stored at 0℃ is added into the pits (31) of the hydrogel microarray (3) to wrap the cell clusters at the bottom of the pits (31), and a culture dish is inverted on the Matrigel to make the bottom of the culture dish contact with the Matrigel, then a constant-temperature heat-conducting block is picked up by a mechanical hand and attached to the outer bottom of the culture dish to keep the constant-temperature heat-conducting block at a constant temperature of 37℃ to assist the Matrigel to solidify; After the solidification is completed, the culture dish is gripped by a mechanical hand, the solidified Matrigel is at the bottom of the culture dish, and the solidified Matrigel is separated from the hydrogel microarray (3); The cell clusters wrapped by the solidified Matrigel in the culture dish retain the original spatial information, so that subsequent organoid culture is continued in the culture dish.

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