Hydrogel micropore array as well as mold and application thereof

Abstract

The invention provides a hydrogel micropore array as well as a mold and application thereof, and belongs to the technical field of cell culture. The hydrogel micropore array and the die thereof provided by the invention can be used for three-dimensional formation and culture of cell microspheres and organoids with controllable quantity, position and size, and further can be used for high-quality high-content imaging. According to the hydrogel micropore array preparation mold provided by the invention, a plurality of hydrogel micropores can be formed in each hole of the micropore plate, so that the repeatability requirement of subsequent further experiments in which cell microspheres are formed is met, and the waste of cells, especially for precious patient-derived cells, is not caused. The number, the position and the size of cell microspheres formed by the hydrogel micropore array manufactured by using the die are controllable; moreover, multiple repeated experiments can be realized only by carrying out one-time treatment, such as drug treatment, in the holes of the cell culture microplate, so that the influence of the operation of an operator on an experiment result is reduced, and the experiment efficiency and accuracy are improved.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, and in particular to a hydrogel microporous array, a mold and an application thereof. Background Art

[0002] The traditional in vitro culture model of animal-derived cells is two-dimensional cell culture. A major problem with using traditional cell culture flasks, culture dishes, and multi-well plates to culture cells is that the cells stay on one plane and grow on the wall. This culture model changes the original three-dimensional growth model in the animal body, lacks various biochemical and mechanical signals that cells can only obtain in a three-dimensional environment, and cannot well simulate the original in vivo growth microenvironment of cells, causing changes in the biological properties and behaviors of the cells themselves. Therefore, the experimental research results obtained by using cells cultured in this model are not convincing.

[0003] Three-dimensional cell culture is closer to the real state of cell growth in vivo. This culture mode can form cell microspheres with three-dimensional structure. In this mode, cells can obtain various biochemical and mechanical signals that cannot be obtained in two-dimensional culture mode through cell-cell and cell-extracellular matrix interactions to maintain normal biological phenotypes. Studies have shown that cell-cell and cell-extracellular matrix interactions are of great significance in regulating cell proliferation, differentiation and migration. Cell microspheres can largely reconstruct the interaction between cells and the external environment, so they can better simulate the microenvironment of cells in vivo, making them closer to cells in vivo in terms of gene expression and function. Three-dimensional cell microspheres have received great attention in the fields of tumor occurrence and development mechanism research, drug screening and bioengineering. There is an urgent need for easy-to-operate, mass-producible, and controllable three-dimensional cell microsphere preparation methods and technologies.

[0004] There are many ways to obtain cell microspheres, including suspension culture using ultra-low adsorption culture dishes or culture plates, hanging drop plates, non-adhesive coating methods, hydrogel microwell array systems, rotating bioreactors, magnetic levitation, and magnetic 3D bioprinting. Although the hanging drop method has advantages in controlling the size and position of cell microspheres, the steps are cumbersome and difficult to prepare in large quantities. Although suspension culture and non-adhesive coating methods can achieve large-scale preparation of cell microspheres, their application is limited by the wide distribution of cell microspheres. The hydrogel microwell array system has a relatively low production cost, and is gradually chosen by most laboratories for its ease of operation over rotating bioreactors, magnetic levitation, and magnetic 3D bioprinting, and its high reuse rate over ultra-low adsorption culture dishes or culture plates and hanging drop plates.

[0005] At present, as time and cost-effectiveness are more valued, high throughput has become a major focus of researchers, including high-throughput imaging technology. Among them, high-content imaging instruments that can meet the needs of high-throughput experiments require matching imaging-specific glass bottom well plates to meet the working requirements of high-content instrument lenses.

[0006] Animal-derived cells are often directly implanted into ultra-low adsorption well plates to form spheres, but the number, position, and size of the cell microspheres formed in the wells cannot be controlled manually. The lack of low adsorption of high-content well plates is not conducive to the formation of cell microspheres, which also hinders the application of high-content imaging in high-throughput imaging of cell microspheres.

[0007] The main means to solve the above problems is to make a hydrogel microwell array system by customizing microsphere culture special well plates and using special positive molds. Special well plates are expensive and have low reuse rates, which cannot meet the current research requirements for high-throughput imaging. For example, SUN Bioscience's Gri3D® microwell plates and STEMCELL's AggreWell™ microwell plates. Commercial special positive molds also have the disadvantage of high cost, such as MicroTissues Inc.'s 3D Petri Dish®. Although self-developed positive molds can solve the problem of high cost, they cannot be well applied to 96-well plates suitable for higher-throughput imaging, such as the molds mentioned in many literatures (1-3); in addition, the process of making hydrogel microwell arrays is time-consuming and requires high operator operation, and has poor reproducibility. The above existing experimental technical solutions are difficult to use for the three-dimensional formation and culture of cell microspheres and organoids with controllable quantity, position and size, and thus difficult to use for high-quality high-content imaging.

[0008] References: 1.Vrij E, Rouwkema J, LaPointe V, van Blitterswijk C, TruckenmüllerR, Rivron N. Directed Assembly and Development of Material-Free Tissues withComplex Architectures. Adv Mater. 2016;28(21):4032-9. 2.Sun 3.Gonzalez-Fernandez T, Tenorio AJ, Leach JK. Three-DimensionalPrinted Stamps for the Fabrication of Patterned Microwells and High-Throughput Production of Homogeneous Cell Spheroids. 3D Print Addit Manuf. 2020;7(3):139-47. Summary of the invention The purpose of the present invention is to provide a hydrogel microwell array and its mold and application, which can be used for the three-dimensional formation and cultivation of cell microspheres and organoids with controllable quantity, position and size, and then for high-quality high-content imaging.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a hydrogel microwell array preparation mold, comprising a base and a plurality of columns connected to the base, wherein a plurality of microcolumns and a plurality of support columns are arranged on the top of each column; The characteristic size of the microcolumns is 0.4-0.6 mm, and the height is 0.7-0.8 mm; The support column has a diameter of 0.7-0.9 mm and a height of 2.5-3 mm.

[0010] Preferably, the support column is located outside the microcolumn.

[0011] Preferably, the distance between the centers of two adjacent microcolumns is 0.8-1.2 mm.

[0012] Preferably, the top view shape of the microcolumn includes a circle, a square, a hexagon, an octagon or a decagon; For circular micropillars, the characteristic dimension refers to the diameter; For square micropillars, the characteristic dimension refers to the side length; For hexagonal, octagonal or decagonal micropillars, the characteristic size refers to the minimum circumscribed circle diameter; The multiple micro-pillars are arranged in a 3*3 array on the top of the pillar.

[0013] Preferably, the height of the column is 14-15 mm; On the base, the distance between the centers of two adjacent columns is 8-10 mm; The multiple columns are arranged in a 6*10 array on the base.

[0014] Preferably, the material of the mold includes resin material, metal material, silicone material or composite material.

[0015] The resin material includes polylactic acid, acrylonitrile-butadiene-styrene copolymer, polyethylene terephthalate, high impact polystyrene, acrylonitrile-styrene-acrylate, polypropylene, polymethyl methacrylate, polyamide, polycarbonate-acrylonitrile-butadiene-styrene, acrylamide resin and the like.

[0016] The present invention also provides a hydrogel micropore array, which is obtained by molding the hydrogel micropore array preparation mold, and the microcolumns in the mold are molded to form array micropores.

[0017] Preferably, the hydrogel material of the hydrogel microporous array includes one or more of agarose, gum arabic, xanthan gum, glucomannan, carrageenan, locust bean gum, alginate, fucoidan, xylan, mannan or galactomannan.

[0018] The present invention also provides the use of the hydrogel microporous array in high-content imaging of cell microspheres or organoids.

[0019] Preferably, the high-content imaging comprises the following steps: Placing cells in array microwells to form cell microspheres or organoids; Perform high-content imaging on the hydrogel microwell array to obtain image results; The cells are cells derived from normal human tissues, human transformed cells, human tumor cells, cells derived from normal animal tissues, animal transformed cells, animal tumor cells, a co-culture system of tumor cells and other types of cells, human primary tumor cells, a co-culture system of human primary tumor cells and other types of cells, or patient-derived xenograft tumor cells.

[0020] Beneficial effects of the present invention: The structure of the microcolumns of the hydrogel microwell array preparation mold provided by the present invention can be selected in different shapes to adapt to the different requirements of different types of cells for physical environments such as mechanical force; the mold can be adapted to cell culture microplates, effectively improving the experimental throughput. The design of the mold allows multiple hydrogel microwells to be formed in each well of the microplate, meeting the repeatability requirements of further experiments subsequent to the formation of cell microspheres therein without causing cell waste, especially for precious patient-derived cells.

[0021] The number, position, and size of cell microspheres formed by the hydrogel microwell array made using the mold are all controllable; and only one treatment, such as drug treatment, is required in the wells of the cell culture microplate to achieve multiple repeated experiments, reducing the impact of the operator's own operations on the experimental results and improving experimental efficiency and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A magnified schematic diagram of the top of the microarray production mold; Figure 2 Schematic diagram of the overall structure of the integrated mold for microarray production; Figure 3 Photographs were taken of single wells of an agarose microwell array molded from a mold; Figure 4 The 10x high-content images of 4T1 tumor cell microspheres cultured in agarose microwell arrays are shown in the figure below. From left to right, they are bright field images, GFP fluorescence images, and bright field and fluorescence merge images. Figure 5 The co-culture microspheres of lymphoma patient-derived xenograft tumor (PDX) cells and MRC5-LB cells were used for 2-fold high-content testing of the anti-tumor drug chidamide. The upper part is the bright field image of the control group and the drug-treated group, and the lower part is the fluorescence Merge image of the control group and the drug-treated group. Each group has 6 replicates. Figure 6 The size distribution statistics of the co-cultured microspheres of lymphoma PDX cells and MRC5-LB cells were taken for high-content bright field photography at cell densities of 1.25E4 and 1.5E4 per well; Figure 7 This is a statistical analysis of the average fluorescence intensity values ​​of different channels in the control group and the drug-treated group of the co-culture microspheres of lymphoma PDX cells and MRC5-LB cells used in the drug sensitivity test of chidamide. DETAILED DESCRIPTION

[0023] The invention provides a hydrogel microwell array preparation mold, comprising a base and a plurality of columns connected to the base, wherein the top of each column is provided with a plurality of microcolumns, a plurality of support columns and a columnar support base; the characteristic size of the microcolumns is 0.4-0.6 mm, and the height is 0.7-0.8 mm; the diameter of the support columns is 0.7-0.9 mm, and the height is 2.5-3 mm; the diameter of the columnar support base is 3.3-3.5 mm, and the height is 1.8-2.2 mm.

[0024] In the present invention, preferably, the microcolumn is located above the cylindrical support base, preferably, the support column is located outside the microcolumn and the cylindrical support base, preferably, the distance between the centers of two adjacent microcolumns is 0.8~1.2mm; the diameter of the cylindrical thin base on which the support column falls is 4.5~5.5mm, and the height of the thin base is 0.8~1.2mm. Preferably, the top view shape of the microcolumn includes a circle, a square, a hexagon, an octagon or a decagon; for a circular microcolumn, the characteristic dimension refers to the diameter; for a square microcolumn, the characteristic dimension refers to the side length; for a hexagonal, octagonal or decagonal microcolumn, the characteristic dimension refers to the minimum circumscribed circle diameter; the multiple microcolumns are arranged in a 3*3 array at the top of the column. Preferably, the height of the column is 14~15mm; on the base, the distance between the centers of two adjacent columns is 8~10mm; the multiple columns are arranged in a 6*10 array on the base.

[0025] Preferably, the material of the mold includes resin materials such as polylactic acid, acrylonitrile-butadiene-styrene copolymer, polyethylene terephthalate, high impact polystyrene, acrylonitrile-styrene-acrylate, polypropylene, polymethyl methacrylate, polyamide, polycarbonate-acrylonitrile-butadiene-styrene, acrylamide resin, metal materials, silicone materials or composite materials. Preferably, the surface of the mold is treated with silanization for anti-sticking.

[0026] The present invention also provides a hydrogel microporous array, which is obtained by molding the above hydrogel microporous array preparation mold, and the microcolumns in the mold are molded to form array micropores. Preferably, the hydrogel material of the hydrogel microporous array includes one or more of agarose, gum arabic, xanthan gum, glucomannan, carrageenan, locust bean gum, alginate, fucoidan, xylan, mannan or galactomannan. Preferably, the molding method comprises the following steps: weighing the dry hydrogel raw material powder, placing it in an autoclaved glass bottle, adding sterile PBS, and rotating to dissolve; transferring the glass bottle to a microwave oven, heating it until the solution boils to completely dissolve the powder, stopping heating every 10 seconds during the process, taking the glass bottle out of the microwave oven and rotating to promote dissolution; sterilizing the glass bottle in a semi-tightened state with high-pressure steam for 30 minutes, tightening it after completion and placing it between cells for standby; placing the autoclaved glass bottle, mold, and dry metal bath in a biological safety cabinet for ultraviolet irradiation for 30 minutes; turning on the water bath between cells and heating it to 80°C for standby, and at the same time turning on the dry metal bath and heating it to 80°C for standby; placing the autoclaved glass bottle in an 80°C water bath to melt the hydrogel; placing a 96-well plate on the dry metal bath, and adding the melted hot hydrogel solution to the wells of the 96-well plate at a volume of 70 μl per well to avoid bubbles when mixing or pipetting the hydrogel solution; gently pressing the mold vertically into the 96-well plate on which the hydrogel is laid on the dry metal bath, and adjusting the temperature of the dry metal bath to 20 ℃, and cool and solidify the hydrogel solution in the 96-well plate for 3 to 5 minutes; after the hydrogel is solidified, place the 96-well plate on the dry metal bath flat on the operating table of the biosafety cabinet, and gently pull out the mold vertically to complete the production of the entire plate of the hydrogel microwell array.

[0027] The present invention also provides the use of the above hydrogel microwell array in high-content imaging of cell microspheres or organoids. Preferably, the high-content imaging comprises the following steps: placing cells in array micropores to form cell microspheres or organoids; performing high-content imaging on the hydrogel microwell array to obtain image results; the cells are cells derived from normal human tissues, human transformed cells, human tumor cells, cells derived from normal animal tissues, animal transformed cells, animal tumor cells, co-culture systems of tumor cells and other types of cells, human primary tumor cells, co-culture systems of human primary tumor cells and other types of cells, or patient-derived xenograft tumor cells. Preferably, the cells derived from normal human tissues include human embryonic lung diploid cells, such as MRC-5, human fetal liver cells, such as CCC-HEL-1, human embryonic kidney cells, such as HEK-293, human embryonic myocardial tissue-derived cells, such as CCC-HEH-2, human embryonic pancreatic tissue-derived cells, such as CCC-HPE-2, human embryonic bladder tissue-derived cells, such as CCC-HB-2, human embryonic intestinal mucosal cells, such as CCC-HIE-2, human embryonic tracheal cells, such as CCC-HBE-2, human umbilical vein endothelial cells, Such as HUVEC, human skin fibroblasts, such as HFF-1, human amniotic membrane cells, such as WISH, human embryonic eye scleral fibroblasts, such as HFSF, human mammary epithelial cells, such as MCF-10A; preferably, the human transformed cells include adenovirus-transformed human embryonic kidney cells, such as 293T, Epstein-Barr virus-transformed human lymphocytes, such as JVM-2, SV40 virus-transformed cells, such as HUVEC-T / T, immortalized epidermal cells, such as HaCaT; preferably, the human tumor cells include human liver cancer cells, human lung cancer cells, human breast cancer cells, Cancer cells, human gastric cancer cells, human colorectal cancer cells, human esophageal cancer cells, human tongue cancer cells, human ovarian cancer cells, human choriocarcinoma cells, human bladder cancer cells, human cervical cancer cells, human prostate cancer cells, human nasopharyngeal cancer cells, human oral cancer cells, human skin cancer cells, human renal cancer cells, human lymphoma cells, human glioma cells, human fibrosarcoma cells, human osteosarcoma cells, human melanoma cells, human brain tumor cells, human leukemia cells, human rhabdomyosarcoma cells, human neuroblastoma cells; preferably, the cells derived from normal animal tissues include animal embryonic fibroblasts Animal transformed cells include SV40 virus transformed cells, such as M-1, Epstein-Barr virus transformed cells, such as B95-8;Preferably, the animal tumor cells include animal mastocytoma cells, such as P815, animal sarcoma cells, such as M5076, animal melanoma cells, such as B16, animal liver cancer cells, such as Hepa1-6, animal gastric cancer cells, such as MFC, animal lymphoma cells, such as YAC-1, animal ascites tumor cells, such as S-180, animal breast cancer cells, such as 4T1, animal dendritic cell sarcoma, such as DCS, animal macrophage tumor cells, such as RAW 264.7, animal testicular interstitial cell tumor cells, such as MLTC-1, animal erythroleukemia cells, such as MEL, animal teratoma cells, such as P19, animal myeloma cells, such as FO, animal brain glioma cells, such as C6, animal pituitary tumor cells, such as GH3, animal adrenal pheochromocytoma cells, such as PC-12; preferably, the human primary tumor cells include primary liver cancer cells, primary lung cancer cells, primary breast cancer cells, primary gastric cancer cells, primary colorectal cancer cells, Primary esophageal cancer cells, primary tongue cancer cells, primary ovarian cancer cells, primary choriocarcinoma cells, primary bladder cancer cells, primary cervical cancer cells, primary prostate cancer cells, primary nasopharyngeal cancer cells, primary oral cancer cells, primary skin cancer cells, primary renal cancer cells, primary lymphoma cells, primary glioma cells, primary fibrosarcoma cells, primary osteosarcoma cells, primary melanoma cells, primary brain tumor cells, primary leukemia cells, primary rhabdomyosarcoma cells, primary neuroblastoma cells. In a specific embodiment of the present invention, the use comprises the following steps:; In the 96-well plate for making the hydrogel microarray, 100 μl of complete culture medium is added to each well, and the culture medium is incubated for 15 minutes or longer to balance the hydrogel; the culture medium is removed and replaced with fresh culture medium, and the culture medium is aspirated from the periphery of the 3*3 gel well array during the removal of the culture medium to avoid damage to the array microwells, and the above balance process is repeated once to complete the balance of the hydrogel; the selected cells are digested with trypsin, and the required number of cells to be seeded is counted and prepared, and the cell amount is selected to be 1.6E4~2.5E5 cells per well, preferably 3.1E4~2E5 cells per well, and most preferably 6.25E4~ 1.25E5 cells, and the volume of cell suspension added to each well is 100μl; most of the culture medium around the hydrogel microwell array is carefully aspirated as described above, and the culture plate is tilted so that as much culture medium as possible in the microwells can be removed; 100μl of the prepared cell suspension is added to each well in a rotating manner from the center of the well to the outside to completely immerse the hydrogel; let it stand for 5 to 10 minutes to ensure that the cells fall evenly into each microwell of the microwell array; the 96-well plate is placed in an incubator for overnight culture to form cell microspheres; high-content imaging of the cell microspheres is performed at 2x and 10x high quality.

[0028] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0029] Example 1 This embodiment provides a method for preparing an agarose microarray.

[0030] 1. The raw materials, consumables and instruments required for the preparation of agarose microarray in this embodiment include: agarose powder, sterile PBS, autoclaved glass bottles, 6*10 microarray production molds (such as Figure 1~2 As shown), 96-well plate, microwave oven, dry metal bath, water bath.

[0031] 2. The specific steps are as follows: (1) Weigh 0.2 g of dry agarose powder on an analytical balance and place it in a 50 ml autoclaved glass bottle. Add 10 ml of sterile PBS to cover the agarose powder and gently rotate horizontally to dissolve as much of the powder as possible.

[0032] (2) Transfer the glass bottle to a microwave oven and heat it until the agarose solution boils and the agarose powder is completely dissolved. Stop heating every 10 seconds during this process, take the glass bottle out of the microwave oven and rotate it to promote the dissolution of the agarose, until you are sure that no small translucent agarose fragments remain.

[0033] (3) Sterilize the glass bottle containing completely dissolved agarose solution or gel with high pressure steam for 30 minutes with the bottle cap half-tightened. After sterilization, tighten the bottle cap and place it between cells for later use.

[0034] (4) Place the autoclaved glass bottle, 6*10 microarray production mold, and dry metal bath in a biosafety cabinet and irradiate with UV light for 30 minutes.

[0035] (5) Turn on the intercellular water bath and heat the water in the water bath to 80°C for use. At the same time, turn on the dry metal bath and heat it to 80°C for use.

[0036] (6) Place the autoclaved glass bottle in an 80°C water bath and heat it to melt the agarose gel in the bottle until no small translucent agarose fragments remain.

[0037] (7) Place the 96-well plate on a dry metal bath and add 70 μl of the melted hot agarose solution into each well of the 96-well plate. Avoid creating bubbles when aspirating or pipetting the agarose solution.

[0038] (8) Gently press the prepared mold vertically into the 96-well plate covered with agarose solution on a dry metal bath.

[0039] (9) Adjust the temperature of the dry metal bath to 20 °C and allow the agarose solution in the 96-well plate pressed into the mold to cool for 3 to 5 minutes to allow the agarose solution to completely solidify.

[0040] (10) After the agarose solidifies, transfer the 96-well plate from the dry metal bath and place it flat on the biosafety cabinet operating table. Gently pull out the mold vertically to complete the production of the entire plate of agarose microwell array. Take a photo of each well. Figure 3 shown.

[0041] Example 2 This embodiment provides a method for preparing and characterizing tumor cell microspheres with controllable quantity, position and size using a prepared agarose microwell array.

[0042] 1. The raw materials, consumables and instruments required for the preparation and characterization of tumor cell spheres in this example include: (1) mouse breast cancer cell line 4T1 with GFP fluorescence, (2) 4T1 tumor cell sphere culture medium preparation: RPMI-1640 culture medium + 10% fetal bovine serum FBS + 1% penicillin-streptomycin P / S, (3) 96-well plate containing agarose microarray, (4) trypsin, (5) cell counting plate, (6) 37°C, 5% CO2 cell culture incubator, and (7) Operetta high-content imaging analysis system.

[0043] 2. The specific steps are as follows: (1) Add 100 μl of RPMI-1640 complete medium to each well of the 96-well plate for making agarose microarrays and incubate in a 37°C, 5% CO2 cell culture incubator for 15 min or longer to equilibrate the agarose gel.

[0044] (2) Remove the equilibration medium and replace it with fresh equilibration medium. During the process of removing the medium, aspirate the medium from the periphery of the 3*3 agarose gel well array to avoid damaging the array micropores. Repeat the equilibration process of step (1) once to complete the equilibration of the agarose gel.

[0045] (3) Trypsinize the well-growing 4T1 cells in the T25 culture flask, count and prepare the required number of 4T1 cells to be seeded, select 1.25E5 cells per well, and prepare 100 μl of cell suspension to be added to each well.

[0046] (4) Carefully aspirate most of the culture medium around the agarose gel microwell array as in step (2) and tilt the culture plate so that as much culture medium as possible can be removed from the microwells.

[0047] (5) Add 100 μl of the prepared cell suspension to each well of the well plate in a rotating manner starting from the center of the well and moving outward to completely submerge the agarose gel.

[0048] (6) Let the plate stand for 5 to 10 minutes to ensure that the 4T1 cells fall evenly into each well of the microwell array.

[0049] (7) The well plate was placed in a 37°C, 5% CO2 incubator for overnight culture to form tumor cell spheres.

[0050] (8) The Operetta high-content imaging analysis system was used to perform high-quality imaging of tumor cell spheroids at 2x and 10x to characterize the tumor cell spheroids.

[0051] 10x high-content photo of 4T1 tumor cell microspheres Figure 4 shown.

[0052] Depend on Figure 4 It can be seen that the number, position and size of cell microspheres formed in the molded agarose microwell array are controllable, and the high-content image quality of bright field and GFP fluorescence is high, which can well meet the imaging needs of various experiments.

[0053] Example 3 This embodiment provides a method for preparing and characterizing co-cultured cell microspheres with controllable quantity, position and size using a prepared agarose microwell array.

[0054] 1. The raw materials, consumables and instruments required for the preparation and characterization of co-cultured cell microspheres in this example include: (1) xenograft tumor (PDX) cells derived from diffuse large B-cell lymphoma (DLBCL) patients, (2) human embryonic lung fibroblast MRC5 (MRC5-LB) cells overexpressing CD40L and BAFF, (3) co-culture complete culture medium: DMEM / F12 culture medium + 1% Glutamax + 10mM HEPES + 1% Pen-Strep + 1% N2 + 10ng / ml EGF + 10ng / ml FGF, (4) 96-well plate containing agarose microarray, (5) trypsin, (6) cell counting chamber, (7) 37°C, 5% CO2 cell culture incubator, and (8) Operetta high-content imaging analysis system.

[0055] 2. The specific steps are as follows: (1) Add 100 μl of complete co-culture medium to each well of the 96-well plate for making agarose microarrays and incubate in a 37°C, 5% CO2 cell culture incubator for 15 min or longer to equilibrate the agarose gel.

[0056] (2) Remove the culture medium and replace it with fresh culture medium. During the process of removing the culture medium, aspirate the culture medium from the periphery of the 3*3 agarose gel well array to avoid damaging the array microwells. Repeat the balancing process of step (1) once to complete the balancing of the agarose gel.

[0057] (3) Trypsinize the well-growing MRC5-LB cells and PDX cells, count and prepare the required number of cells for seeding. The amount of cells is 1.5E5 per well. PDX cells and MRC5-LB cells are added in a ratio of 9:1, and 100 μl of cell suspension is prepared for each well.

[0058] (4) Carefully aspirate most of the culture medium around the agarose gel microwell array as in step (2) and tilt the culture plate so that as much culture medium as possible can be removed from the microwells.

[0059] (5) Add 100 μl of the prepared cell suspension to each well of the well plate in a rotating manner starting from the center of the well and moving outward to completely submerge the agarose gel.

[0060] (6) Let the plate stand for 5 to 10 minutes to ensure that the co-cultured cells fall evenly into each microwell of the microwell array.

[0061] (7) Place the well plate in a 37°C, 5% CO2 incubator for overnight culture to form co-cultured cell spheres of PDX cells and MRC5-LB cells.

[0062] Example 4 This embodiment provides an operation and characterization method for drug screening using co-cultured cell spheroids cultured in agarose microwell arrays.

[0063] 1. Agarose Microarray Preparation Microarray plate: Ultra-thin glass bottom 96-well plate (Cellvis P96-1.5HN); Complete Lymphoma Organoid Medium Recipe: DMEM / F12 + 10% PBS + 1% PS + 10 mM HEPES + GlutaMax + 10 ng / mL IL-4; Agarose microarray was prepared as described above: Prepare 1-2 days in advance, incubate with complete medium, and store at 37°C 5% CO2; 2. Cell Preparation MRC5-LB cell preparation: MRC5-LB cells: Human embryonic lung cells stably transfected with lentivirus expressing CD40 ligand (CD40 L), B-cell activating factor (BAFF), and green fluorescent protein (GFP); MRC5-LB cell recovery and culture: Resuscitate MRC5-LB cells 3-5 days in advance to two 6 cm dishes. MRC5-LB cell culture medium formula: DMEM + 10% FBS + 1% PS, culture conditions: 37°C 5% CO2; MRC5-LB cell collection and counting: MRC5-LB cells were collected by trypsin digestion, centrifuged at 250 g for 5 min, the supernatant was discarded, and the cells were resuspended in complete medium and counted; DLBCL PDX Cell Preparation: DLBCL PDX Cell Recovery: The frozen DLBCL PDX cells were taken out of liquid nitrogen, placed in a 37°C water bath to quickly rewarm, and 5 ml of complete medium was added and centrifuged at 400 g for 5 minutes, and the supernatant was discarded; DLBCL PDX cell count: Resuspend the cells in complete medium and count them to prepare approximately 4E6 PDX cells; Prepare mixed cell suspension: Calculation of total number of cells required: Inoculate 12 wells, with a total of 1.5E5 cells in each well, for a total of 2E6 cells. Preparation of mixed cell suspension: DLBCL PDX cells: MRC5-LB cells = 9:1, i.e. 1.8E6 DLBCL PDX cells and 2E5 MRC5-LB cells, prepared into a mixed cell suspension with a cell density of 1.5E6 cells / ml; 3. Cell seeding and observation Cell seeding: Aspirate the complete medium in the well plate, add 100 μl of mixed cell suspension (containing 1.5E5 cells) to each well, and let stand for 15-20 minutes; Observation and cultivation: Observe the cell sedimentation under the microscope and transfer to a 37°C 5% CO2 incubator for culture; Observation of ball formation: The day after inoculation, observe the sphering of co-cultured cells in the microplate under a microscope, and observe the bright field (BF) and FITC channels; 4. Drug responsiveness testing of organoids cultured in microplates Preparation of drug solution: Chidamide was selected as the screening drug, and the drug concentration in the experimental group was 10 μM; Since each well originally contained 100 μl of complete medium, a 2× drug solution was prepared, specifically: 1.4 μl of 10 mM chidamide + 698.6 μl of complete medium, to prepare a drug solution with a final concentration of 20 μM and a total volume of 700 μl; Group design: The groups included an experimental group (test) and a control group (control), with 6 replicates in each group; Drug administration: In the experimental group, 100 μl of the 2× drug solution prepared above was added to each well, and in the control group, 100 μl of complete culture medium was added to each well; Drug reaction observation: 48 h after drug administration, organoids in microplates were photographed using a high-content imaging analyzer (Perkin Elmer Operetta); Dyeing treatment: Dye solution formula: 1.5μM PI dye + 20μM Hoechst-33342 dye + PBS; Aspirate the original culture medium, add 50 μl of dye solution to each well, and directly put it on the machine for subsequent shooting; On-machine operation: Plate selection: Cellvis P96-1.5HN; Magnification: 2x; Environmental settings: 37℃ 5%CO2; Channel settings: bright field BF, GFP (ch2), Hoechst (ch3), PI (ch4); Z stack: 8 × 20 μm; Data processing: ImageJ: Process the bright field and fluorescence images captured by the high-content imaging analyzer to derive the area and mean fluorescence intensity (grayscale value) of the organoid spheres; Graph Pad Prism: The above exported data were analyzed using software and unpaired T test was performed, and a P value less than 0.05 was defined as a significant difference; The results are as follows Figure 5 As shown. Lymphoma PDX cells and MRC5-LB cells were seeded into each well with a total cell number of 1.5E5, and drug treatment was performed after 3 days of culture. High-content imaging was performed 3 days after drug administration. Figure 5 (Top) are bright field images of the control group and the drug-treated group. Figure 5 (Bottom) is the fluorescence merge diagram of the control group and the drug-treated group. The fluorescence intensity and area of ​​the corresponding channel of PI in the drug-treated group were significantly stronger than those in the control group. Figure 6 The sizes of the cell microspheres in the microwells are shown, ranging from 150 μm to 450 μm at a cell density of 1.25E4 per well, and from 200 μm to 450 μm at a cell density of 1.5E4 per well, with high uniformity.

[0064] The data processing results are as follows Figure 7 The results showed that the average fluorescence intensity values ​​of different channels in the control group and the drug-treated group were statistically analyzed, and an unpaired T test was performed. Within the 95% confidence interval, Figure 7 (Left) P=0.1115, Figure 7 (middle) P=0.0010, Figure 7 (right) P=0.0098, indicating that the treatment with cedamide had no significant effect on the growth of MRC5-LB cells, but cedamide significantly inhibited the growth of lymphoma PDX cells, leading to significant death of DLBCL cells. The above results all demonstrate the significant superiority of the hydrogel microporous array of the present invention in the application of cell microsphere drug screening and the high reliability of the results.

[0065] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A hydrogel microwell array preparation mold, characterized in that: It includes a base and a plurality of columns connected to the base, and a plurality of micro columns and a plurality of support columns are arranged on the top of each column; The characteristic size of the microcolumns is 0.4-0.6 mm, and the height is 0.7-0.8 mm; The support column has a diameter of 0.7-0.9 mm and a height of 2.5-3 mm.

2. The hydrogel microwell array preparation mold according to claim 1, characterized in that: The support column is located at the periphery of the microcolumn.

3. The hydrogel microwell array preparation mold according to claim 1, characterized in that: The distance between the centers of two adjacent microcolumns is 0.8~1.2mm.

4. The hydrogel microwell array preparation mold according to claim 1, characterized in that: The top view shape of the microcolumn includes a circle, a square, a hexagon, an octagon or a decagon; For circular micropillars, the characteristic dimension refers to the diameter; For square micropillars, the characteristic dimension refers to the side length; For hexagonal, octagonal or decagonal micropillars, the characteristic size refers to the minimum circumscribed circle diameter; The multiple micro-pillars are arranged in a 3*3 array on the top of the pillar.

5. The hydrogel microwell array preparation mold according to claim 1, characterized in that: The height of the column is 14-15 mm; On the base, the distance between the centers of two adjacent columns is 8-10 mm; The multiple columns are arranged in a 6*10 array on the base.

6. The hydrogel microwell array preparation mold according to any one of claims 1 to 5, characterized in that: The material of the mold includes resin material, metal material, organic silicon material or composite material.

7. A hydrogel microwell array, characterized in that: The hydrogel micropore array is prepared by molding with the mold according to any one of claims 1 to 6, wherein the microcolumns in the mold are molded to form array micropores.

8. The hydrogel microwell array according to claim 7, characterized in that: The hydrogel material of the hydrogel microporous array includes one or more of agarose, gum arabic, xanthan gum, glucomannan, carrageenan, locust bean gum, alginate, fucoidan, xylan, mannan or galactomannan.

9. Use of the hydrogel microwell array according to claim 7 in high-content imaging of cell microspheres or organoids.

10. The use according to claim 9, characterized in that: The high-content imaging comprises the following steps: Placing cells in array microwells to form cell microspheres or organoids; Perform high-content imaging on the hydrogel microwell array to obtain image results; The cells are cells derived from normal human tissues, human transformed cells, human tumor cells, cells derived from normal animal tissues, animal transformed cells, animal tumor cells, a co-culture system of tumor cells and other types of cells, human primary tumor cells, a co-culture system of human primary tumor cells and other types of cells, or patient-derived xenograft tumor cells.

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