Preparation method and application of cell-loaded porous 3D scaffold

Abstract

The invention discloses a preparation method and application of a cell-loaded porous 3D scaffold. The preparation method comprises the following steps: S1, preparing printing ink; s2, the printing ink prepared in the step S1 is added into 3D printing equipment, and a 3D support is printed according to a designed model; the printing ink comprises a main material, an auxiliary material and a cell-loaded suspension, the carrier is applied to dynamic and static culture, testing or screening of carrier cells. The specific surface area of the 3D scaffold is increased to the maximum extent, the dynamic and static culture, test and screening functions of the carrier cells are realized, and the scientific research efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the field of medical model manufacturing and application, and in particular to a preparation method of a cell-laden porous 3D scaffold and its application. Background Art

[0002] The cultivation of mainstream tumor organoids in the world now is mainly culture plate cultivation, and culture plate cultivation generally uses matrigel matrix glue and culture medium containing specific growth factors to carry out static cultivation for the 3D cell mass formed, and this culture method needs to manually change the culture medium regularly, and dynamic cultivation can not be realized, and because the 3D cell mass is a solid structure, the specific surface area is low, the contact area between the 3D cell mass and the culture medium is small, and the exchange is insufficient, and the growth effect of the internal organoid of the 3D cell mass is not good. In addition, although matrigel matrix glue has good compatibility, it is difficult to fully contact with the screening object and the test object in the external world after the organoid in the 3D cell mass is cultivated by the embedding method, and the scientific research activities such as drug screening and detection cannot be effectively carried out. Therefore, how to solve the above-mentioned technical problems is the direction that those skilled in the art need to work hard. Summary of the invention

[0003] The purpose of the present invention is to provide a method for preparing a cell-laden porous 3D scaffold and its application. By using this method and application, it is possible to provide a 3D environment for tumor cell culture, and at the same time, it is possible to provide a multi-material complex porous structure for co-culture of tumor and immune cells, maximize the specific surface area of ​​the 3D scaffold, realize dynamic and static culture, testing and screening functions of cells, and improve scientific research efficiency.

[0004] To achieve the above object, the technical solution adopted by the present invention is: a method for preparing a cell-laden porous 3D scaffold, the steps of which are:

[0005] S1. preparing printing ink;

[0006] S2, adding the printing ink prepared in step S1 into a 3D printing device, and printing a 3D bracket according to the designed model;

[0007] The printing ink comprises a main material, an auxiliary material and a cell suspension.

[0008] In the above technical solution, the main material includes one or more mixtures of photosensitive materials and non-photosensitive materials;

[0009] The photosensitive material comprises one or more of methacryloylated collagen, methacryloylated gelatin, methacryloylated hyaluronic acid, methacryloylated silk protein, methacryloylated elastin, methacryloylated heparin, methacryloylated keratin, methacryloylated sericin, methacryloylated polyvinyl alcohol, methacryloylated polycaprolactone, methacryloylated chitosan, methacryloylated dextran, methacryloylated sodium alginate, methacryloylated chondroitin sulfate, polyether F 127 diacrylate, and polyethylene glycol diacrylate;

[0010] The non-photosensitive material includes one or more of collagen, gelatin, silk fibroin, keratin, elastin, sodium alginate, chitosan, agarose, polyvinyl alcohol, polycaprolactone, polyurethane, polyethylene glycol, hydroxyapatite, tricalcium phosphate, polyether F127, porcine, bovine, mouse or human decellularized matrix, and nanoclay;

[0011] The cell suspension contains cells and a culture medium solution suitable for the cells being loaded;

[0012] And / or, the cells in the cell suspension include one or more of a tumor organoid cell suspension extracted from cancerous tissue after subculture, tumor cell line cells, extracted tumor fibroblasts, extracted tumor stem cells, immune cells, and vascular endothelial cells;

[0013] And / or, the cells contained in the cell suspension also include one or more of organoid cell suspensions extracted from normal tissues and cultured after subculture, tissue cell lines, stem cells extracted from tissues, iPSCs and functional cells differentiated from iPSCs, embryonic stem cells and functional cells differentiated from them, immune cells, and vascular endothelial cells.

[0014] In the above technical solution, the mass ratio of the photosensitive material in the printing ink is 0.1% to 90%, the mass ratio of the non-photosensitive material in the printing ink is 0.1% to 80%, the mass ratio of the photoinitiator in the printing ink is 0.1% to 10%; the mass ratio of the auxiliary material in the printing ink is 0% to 50%; the cell content in the cell suspension is 1*10 3 ~1*10 9 .

[0015] In the above technical solution, the 3D printing device is a light-curing 3D printer or an extrusion direct writing 3D printer;

[0016] When the 3D printing device adopts a light-curing 3D printer, the auxiliary materials include a photoinitiator and a light absorber;

[0017] When the 3D printing device adopts an extrusion direct writing 3D printer, the auxiliary material includes a thickener;

[0018] And / or, the photoinitiator includes one or more of Irgacure 2959, phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP), lithium phosphate, TPO, proanthocyanidin, camphorquinone, eosin Y, terpyridine ruthenium chloride and sodium persulfate, dihydro-4,4-dimethyl-2,3-furandione, ethyl pyruvate, and lipoic acid; the light absorber includes one or more of tartrazine, rutin, phenol red, eosin, phthalocyanine blue, curcumin, and brilliant blue;

[0019] And / or, the thickener includes one or more of carboxymethyl cellulose, polyether F127, lithium magnesium silicate, sodium alginate, chitosan, agarose, and methacrylated gelatin.

[0020] In the above technical solution, in step S2, the designed model is the first model or the second model, the 3D bracket printed according to the first model is a porous 3D bracket, and the 3D bracket printed according to the second model is a porous 3D bracket with an inlet and an outlet;

[0021] The 3D scaffold printed according to the first model has a plurality of interconnected holes, and the 3D scaffold contains cells, and the cells can grow in the 3D scaffold;

[0022] After step S2, the 3D scaffold is placed in a culture dish or culture plate, and after adding culture medium, it is placed in a carbon dioxide incubator for culture, and the culture medium is replaced regularly to provide a static culture environment for the 3D scaffold;

[0023] The 3D scaffold printed according to the second model has a sealing structure, an inlet and an outlet connected to the sealing structure, the sealing structure has a plurality of holes connected to each other, the inlet and the outlet are respectively connected to the holes in the sealing structure, and the 3D scaffold contains cells, and the cells can grow in the 3D scaffold;

[0024] After the step S2, the hoses are connected to the inlet and outlet pre-printed on the 3D scaffold respectively, the pump is connected to the reservoir through the hose, the pump, the hose, the reservoir and the 3D scaffold are placed in a carbon dioxide incubator, and the pump is turned on. The pump pumps the culture medium in the reservoir into the inlet of the 3D scaffold through the hose, and the culture medium flows out from the outlet of the 3D scaffold after passing through the interconnected holes in the 3D scaffold, thereby realizing the dynamic flow of the culture medium in the molded 3D scaffold and providing a dynamic culture environment for the 3D scaffold;

[0025] And / or, after the cell culture is completed, the culture medium is replaced with a test substance or a screening substance to provide a testing or screening environment for the 3D scaffold.

[0026] In the above technical solution, the 3D bracket has a porous grid structure interconnected with each other, the diameter of the grid is 0.1mm to 5mm, and the wall thickness of the grid is 0.05mm to 3mm.

[0027] The present invention also provides a cell-laden porous 3D scaffold, which is prepared by using the above-mentioned method for preparing the cell-laden porous 3D scaffold.

[0028] The present invention also provides an application method of a cell-laden porous 3D scaffold, comprising a 3D scaffold, wherein the 3D scaffold comprises a porous 3D scaffold, or the 3D scaffold is a porous 3D scaffold with an inlet and an outlet, and the 3D scaffold is prepared by the above-mentioned preparation method of the cell-laden porous 3D scaffold;

[0029] When the 3D scaffold is a porous 3D scaffold, the 3D scaffold is placed in a culture dish or a culture plate, and then the culture medium, the test object or the screening object is added to the culture dish or the culture plate, and the culture dish or the culture plate is placed in a carbon dioxide incubator to achieve a static culture, test or screening environment for the 3D scaffold;

[0030] When the 3D scaffold is a 3D porous scaffold with an inlet and an outlet, the hoses are respectively connected to the inlet and outlet pre-printed on the 3D scaffold, the pump is connected to the reservoir through the hose, culture medium, test material or screening material is added to the reservoir, the pump, hose, reservoir and 3D scaffold are placed in a carbon dioxide incubator and the pump is turned on. The pump pumps the culture medium, test material or screening material in the reservoir into the inlet of the 3D scaffold through the hose, and the culture medium, test material or screening material flows out from the outlet of the 3D scaffold after passing through the interconnected pores in the 3D scaffold, thereby realizing dynamic culture, dynamic testing or dynamic screening of the 3D scaffold.

[0031] In the above technical solution, the test substance is one or more of small molecule chemical drugs, albumin conjugates and liposomes of small molecule chemical drugs, radioactive small molecule chemical drugs, macromolecular protein drugs, nucleic acid drugs, nanoparticle drugs or cell therapy drugs.

[0032] In the above technical scheme, the screening object is one or more of peripheral blood mononuclear cells, universal T cells, NK cells, macrophages differentiated from iPSC, stem cells extracted from tissues, tumor-infiltrating T cells, CAR-modified T cells or NK cells.

[0033] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0034] 1. The present invention adopts 3D printed cell-loaded porous scaffolds, which can effectively solve the problem of low specific surface area of ​​3D cell clusters formed by matrigel gel embedding and sufficient contact between organoids and external screening and test objects, so that after culture, they can be easily and fully contacted with external screening and test objects, thereby improving the efficiency and effect of drug screening and testing;

[0035] 2. In the present invention, tumor cells, PDO suspension, tissue cells, stem cells and / or immune cells can be printed into porous scaffolds with a large specific surface area. Organoids are embedded in the grid wall of the porous scaffolds. The 3D environment and large contact area improve the culture efficiency. It can be applied to the fields of dynamic cell culture, drug effectiveness, sensitivity detection and drug screening. At the same time, it can provide immune cells with a tumor-like environment to stimulate or enhance the ability of immune cells to recognize and kill tumors.

[0036] 3. In the present invention, the cell suspension is directly mixed into the printing ink for 3D printing, so that the printed 3D scaffold has cells, which can flexibly realize the multi-material complex structure of multi-cell co-culture, and imitate the microenvironment suitable for the growth of different cells to the maximum extent. At the same time, the inlet and outlet structures are constructed in the structure, so as to realize the dynamic culture of cells and the testing functions such as drug screening and drug sensitivity detection, and effectively improve the efficiency of scientific research;

[0037] 4. The present invention adopts 3D printing to print out a 3D scaffold with cells mixed inside. Compared with the existing organoid chips, it improves the convenience of production, reduces the production cost, and can produce 3D porous scaffolds with more complex structures. It can also improve the scalability of scientific research, and the cells are directly mixed into the printing ink, so that the cells can be evenly distributed in the 3D scaffold, which can improve the culture effect and convenience of the cells, and can also improve the effect and convenience of drug screening and testing of test objects and screened objects;

[0038] 5. The present invention can adopt light-curing 3D printing or extrusion direct writing 3D printing, and the printing method can be selected, and the printing application range is wide;

[0039] 6. The 3D bracket printed in the present invention may have an inlet and outlet, or may not have an inlet and outlet, and may be applied in different ways and selected according to different application occasions. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is a schematic diagram of the structure of the 3D bracket in the second embodiment of the present invention;

[0041] Figure 2 This is a picture of colorectal cancer organoids grown in a 3D scaffold in Example 2 of the present invention;

[0042] Figure 3 is a schematic diagram of the structure of the 3D bracket in the third embodiment of the present invention;

[0043] Figure 4 It is a schematic diagram of the structure of the 3D bracket in the fourth embodiment of the present invention.

[0044] Among them: 1. Import; 2. Export. DETAILED DESCRIPTION

[0045] The present invention will be further described below in conjunction with the accompanying drawings and embodiments:

[0046] Embodiment 1: A method for preparing a cell-laden porous 3D scaffold, the steps of which are:

[0047] S1. preparing printing ink;

[0048] S2, adding the printing ink prepared in step S1 into a 3D printing device, and printing a 3D bracket according to the designed model;

[0049] The printing ink comprises a main material, an auxiliary material and a cell suspension.

[0050] In this embodiment, the main material, auxiliary material and cell suspension are evenly mixed to prepare printing ink, which is placed in a 3D printing device to print a 3D scaffold according to the design model. The cell suspension contains cells that need to be subsequently cultured, tested or screened, and the 3D scaffold is designed according to the required application environment. In this way, the cells can fully contact with the culture medium, screening objects or test objects, thereby improving the efficiency and effectiveness of culture, testing and screening.

[0051] Wherein, the main material includes one or more mixtures of photosensitive materials and non-photosensitive materials;

[0052] The photosensitive material includes one or more of methacrylated collagen, methacrylated gelatin, methacrylated hyaluronic acid, methacrylated silk protein, methacrylated elastin, methacrylated heparin, methacrylated keratin, methacrylated sericin, methacrylated polyvinyl alcohol, methacrylated polycaprolactone, methacrylated chitosan, methacrylated dextran, methacrylated sodium alginate, methacrylated chondroitin sulfate, polyether F 127 diacrylate, and polyethylene glycol diacrylate.

[0053] Among them, photosensitive materials are the main materials for photocuring printing. They have the property of changing rapidly from liquid to solid under light excitation within a few seconds. The 3D printer solidifies the area to be cured after letting light pass through it, and then solidifies it layer by layer (in the process of printing the 3D bracket through the 3D printer, each layer of printing ink is printed by light, and the thickness of each layer is generally about 0.05mm), thereby realizing a complex printing mechanism.

[0054] Among them, the photocuring 3D printer mainly includes a digital light processing technology (DLP) 3D printer with DMD optical projection technology, a liquid crystal display technology (LCD) 3D printer and a stereolithography modeling technology (SLA) SLA 3D printer using a laser point light source.

[0055] The non-photosensitive material includes one or more of collagen, gelatin, silk fibroin, keratin, elastin, sodium alginate, chitosan, agarose, polyvinyl alcohol, polycaprolactone, polyurethane, polyethylene glycol, hydroxyapatite, tricalcium phosphate, polyether F127, decellularized matrix of pig, cow, mouse or human origin, and nanoclay.

[0056] Non-photosensitive materials are mainly used to provide different cells with the microenvironment required for growth, or to adjust the mechanical properties of the material after 3D scaffold printing, or they also contain factors that are beneficial to the generation of cells, thereby enhancing the culture environment of cells.

[0057] The cells in the cell suspension include one or more of a tumor organoid cell suspension extracted from cancerous tissue after subculture, tumor cell line cells, extracted tumor fibroblasts, extracted tumor stem cells, immune cells, and vascular endothelial cells;

[0058] And / or, the cells in the cell suspension also include one or more of organoid cell suspension extracted from normal tissue and cultured, tissue cell lines, stem cells extracted from tissue, iPSCs and functional cells differentiated from iPSCs, immune cells, and vascular endothelial cells.

[0059] The cells in the cell suspension are the cells required to be cultured, tested or used for screening the screening objects. According to the cells that need to be cultured, the cell suspension corresponding to the cells required to be cultured, tested or used for screening the screening objects is mixed into the printing ink, and then the corresponding 3D scaffold is printed according to the cells required to be cultured, tested or used for screening the screening objects, which is used for cell culture, testing or screening of the screening objects under the cell microenvironment.

[0060] Wherein, the 3D printing device is a light-curing 3D printer or an extrusion direct writing 3D printer.

[0061] When the 3D printing device adopts a light-curing 3D printer, the auxiliary materials include a photoinitiator and a light absorber;

[0062] The photoinitiator includes one or more of Irgacure 2959, phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP), lithium phosphate, TPO, proanthocyanidin, camphorquinone, eosin Y, terpyridine ruthenium chloride and sodium persulfate, dihydro-4,4-dimethyl-2,3-furandione, ethyl pyruvate, and lipoic acid; the light absorber includes one or more of tartrazine, rutin, phenol red, eosin, phthalocyanine blue, curcumin, and brilliant blue.

[0063] In 3D printing, photoinitiators initiate monomer polymerization, cross-linking and curing through light exposure; light absorbers are used to improve printing accuracy, prevent excessive cross-linking, and solve the problem of over-curing caused by light scattering.

[0064] When the 3D printing device adopts an extrusion direct writing 3D printer, the auxiliary material includes a thickener;

[0065] The thickener includes one or more of carboxymethyl cellulose, polyether F127, lithium magnesium silicate, sodium alginate, chitosan, agarose, and methacrylated gelatin. The thickener thickens the printing ink so that it can be extruded into filaments for printing.

[0066] Among them, the extrusion direct writing 3D printer mainly includes an air pump extrusion 3D printer that uses compressed air as power to blow the bio-ink out of the material tube, a motor screw extrusion 3D printer that uses a motor to drive a screw to compress the bio-ink in the material tube for extrusion, and a DIW (Direct Ink Writing) 3D printer that extrude the bio-ink from a micro-nozzle through a nozzle similar to an inkjet printer and deposits it into a customized pattern under the movement of the printing substrate.

[0067] Furthermore, extrusion direct writing printing is also suitable for light-curing printing materials. Therefore, when the 3D printing equipment uses an extrusion direct writing 3D printer, the auxiliary materials include a photoinitiator in addition to a thickener. After the material is extruded and formed, it can be cured by light for a secondary cure to improve the strength of the 3D scaffold.

[0068] The mass ratio of the photosensitive material in the printing ink is 0.1% to 90%, the mass ratio of the non-photosensitive material in the printing ink is 0.1% to 80%, the mass ratio of the photoinitiator in the printing ink is 0.1% to 10%; the mass ratio of the light absorber in the printing ink is 0% to 0.5%; the mass ratio of the thickener in the printing ink is 0% to 50%; the cell content in the cell suspension is 1*10 3 ~1*10 9 .

[0069] Wherein, in the present invention, in the step S2, the designed model is the first model or the second model, the 3D bracket printed according to the first model is a porous 3D bracket, and the 3D bracket printed according to the second model is a porous 3D bracket with an inlet and an outlet;

[0070] The 3D scaffold printed according to the first model has a plurality of interconnected holes, and the 3D scaffold contains cells, and the cells can grow in the 3D scaffold;

[0071] After step S2, the 3D scaffold is placed in a culture dish or culture plate, and after adding culture medium, it is placed in a carbon dioxide incubator for culture, and the culture medium is replaced regularly to provide a static culture environment for the 3D scaffold. After the culture is completed, the culture medium can be replaced with a test object or a screening object for testing the cell-carrying cells or screening the screening object to provide a static test or screening environment for the 3D scaffold.

[0072] In this method, there are interconnected holes on the 3D scaffold, and the cells are placed in a 3D scaffold with a porous structure. This can effectively solve the problem of low specific surface area of ​​3D cell clumps formed by matrigel gel embedding and sufficient contact between organoids and external screening and test objects, and a static culture, testing or screening environment is adopted.

[0073] The 3D scaffold printed according to the second model has a sealing structure, an inlet and an outlet connected to the sealing structure, the sealing structure has a plurality of holes connected to each other, the inlet and the outlet are respectively connected to the holes in the sealing structure, and the 3D scaffold has cells therein, and the cells can grow in the 3D scaffold;

[0074] After the step S2, the hose is connected to the inlet and outlet pre-printed on the 3D scaffold, the pump is connected to the reservoir through the hose, the pump, the hose, the reservoir and the 3D scaffold are placed in a carbon dioxide incubator, and the pump is turned on. The pump pumps the culture medium in the reservoir into the inlet of the 3D scaffold through the hose, and then flows out from the outlet of the 3D scaffold through the interconnected holes in the 3D scaffold, so as to realize the dynamic flow of the culture medium in the molded 3D scaffold and provide a dynamic culture environment for the 3D scaffold. The pump is a peristaltic pump, an air pump or a syringe pump.

[0075] After the culture is completed, the culture medium can be replaced with test substances or screening substances for testing the loaded cells or screening the screening substances, so as to provide a dynamic testing or screening environment for the 3D scaffold.

[0076] The two 3D scaffolds are used to provide static / dynamic culture, testing or screening environments for cells according to different cell carriers.

[0077] The 3D bracket has a porous grid structure interconnected with each other, the diameter of the grid is 0.1 mm to 5 mm, and the wall thickness of the grid is 0.05 mm to 3 mm.

[0078] In the present invention, the cell carrier can be printed into a porous scaffold with a large specific surface area, and the organoids are embedded in the grid wall of the porous scaffold. The culture efficiency is improved through the 3D environment and the large contact area. It can be applied to dynamic and static cell culture, drug effectiveness, sensitivity detection and drug screening. At the same time, it can provide immune cells (immune cells are used as an example of screening objects) with a tumor-like environment to stimulate or enhance the ability of immune cells to recognize and kill tumors.

[0079] The present invention further provides a cell-laden porous 3D scaffold, which is prepared by the above-mentioned method for preparing the cell-laden porous 3D scaffold.

[0080] The 3D scaffold includes a porous 3D scaffold, see Figure 1 As shown, the 3D scaffold is mainly used for static culture, testing or screening of cells.

[0081] Or the 3D scaffold is a porous 3D scaffold with an inlet and an outlet, see Figure 4 As shown, the 3D scaffold is mainly used for dynamic culture, testing or screening of cells to improve scientific research efficiency.

[0082] The present invention also provides an application method of a cell-carrying porous 3D scaffold. When the 3D scaffold is a porous 3D scaffold, the 3D scaffold is placed in a culture dish or a culture plate, and then a culture medium, a test object or a screening object is added to the culture dish or the culture plate, and the culture dish or the culture plate is placed in a carbon dioxide incubator to achieve a static culture, test or screening environment for the 3D scaffold.

[0083] When the 3D scaffold is a 3D porous scaffold with an inlet and an outlet, the hoses are respectively connected to the inlet and outlet pre-printed on the 3D scaffold, the pump is connected to the reservoir through the hose, the culture medium, the test object or the screened object are added to the reservoir in sequence, the pump, the hose, the reservoir and the 3D scaffold are placed in a carbon dioxide incubator together and the pump is turned on, the pump pumps the culture medium, the test object or the screened object in the reservoir into the inlet of the 3D scaffold through the hose, and the culture medium, the test object or the screened object flows out from the outlet of the 3D scaffold after passing through the interconnected pores in the 3D scaffold, thereby realizing dynamic culture, dynamic testing or dynamic screening of the 3D scaffold and improving scientific research efficiency.

[0084] Wherein, the test substance is one or more of small molecule chemical drugs, albumin conjugates and liposomes of small molecule chemical drugs, radioactive small molecule chemical drugs, macromolecular protein drugs, nucleic acid drugs, nanoparticle drugs or cell therapy drugs.

[0085] Among them, small molecule chemical drugs mainly include I 131 , nab-paclitaxel, liposomal doxorubicin, doxycycline, or oxaliplatin;

[0086] Nucleic acid drugs mainly include one or more of siRNA, mRNA liposomes, c ircRNA or Lnc-RNA;

[0087] Nanoparticle drugs mainly include one or more of gold nanoparticles, silver nanoparticles, and iron-containing magnetic nanoparticles;

[0088] Cell therapy drugs mainly include one or more of CAR-T, TCR-T, CAR-NK, TIL, immune cell populations processed after self-extraction, macrophages, universal T cell differentiated from iPSC, NK cell, macrophages or stem cells.

[0089] The screening object is one or more of peripheral blood mononuclear cells, universal T cells differentiated from iPSC, NK cells, macrophages, stem cells extracted from tissues, tumor-infiltrating T cells, CAR-modified T cells or NK cells.

[0090] Example 2: A method for preparing a cell-laden porous 3D scaffold and its application. In this example, it is used to culture colorectal cancer organoids. It adopts a photocuring printing method, and the steps are as follows:

[0091] A1. Design of porous 3D scaffold model: Figure 1As shown, a porous cylindrical model is designed based on CAD software, with a cylindrical diameter of 12.50 mm, a thickness of 2.30 mm, a pore size of 1.2 mm-1.5 mm, and a wall thickness of 0.4-0.8 mm. The model is imported into the software supporting the light-curing 3D printer, and a printable executable file is generated after setting;

[0092] A2. Turn on the light-curing 3D printer, set the temperature to 37°C to preheat the printer chamber, and import the model designed in step A1 into the light-curing 3D printer;

[0093] A3. Prepare printing ink: prepare RPMI1640 solution of 15% methacryloyl gelatin (GelMA), 1.5% methacryloyl hyaluronic acid (HAMA), 0.03% tartrazine, and 0.75% phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP) (RPMI 1640 solution is used to dissolve relevant printing ink and photoinitiator, which can make the carried cells survive better in the 3D scaffold), and then filter and sterilize with a 0.22um filter membrane to prepare a mixed solution 1 for mixing with the cell suspension;

[0094] A4. Preparation of colorectal cancer PDO (cell suspension): Take several rice-sized tumor tissues or PDO tissues after passage, wash them several times with pre-cooled phosphate buffered saline (PBS) containing 1× penicillin-streptomycin solution; cut the tissue into minced meat (PDO tissue does not need to be cut); transfer the minced tissue to a centrifuge tube, add an appropriate amount of RPMI containing 3 mg / mL of collagenase IV (type IV collagenase) and 5 MU / mL of DNase I (deoxyribonuclease I) 1640 medium, shake and digest at 37℃ for 1 hour; filter the fully lysed cells or cell tissue suspension with 100μm and 40μm cell filters to remove residual tissue; centrifuge the filtrate at 1500rpm for 5 minutes, remove the supernatant; then use red blood cell lysis buffer to lyse the red blood cells for 5 minutes; add DMEM medium to the obtained single cell suspension, which contains Wnt pathway agonist, MAPK / P38 inhibitor, TGFβ pathway inhibitor, ROCK inhibitor, EGF, bFGF and penicillin-streptomycin, and the cell concentration is 1*10 7 ~1*10 8 , used for subsequent mixing with mixed solution 1;

[0095] A5. Mix the mixed solution 1 and the single cell suspension in a volume ratio of 2:1, mix well and place in the loading tank of the preheated light-curing 3D printer, call out the printable execution file to print the cell-loaded porous 3D scaffold;

[0096] A6. After the 3D bracket is printed, take out the porous 3D bracket on the stage (the printed 3D bracket is consistent with the designed model, such as Figure 1 After washing with PBS, the cells were placed in a well plate and cultured with colorectal cancer organoid induction medium.

[0097] like Figure 2 Shown is a picture of colorectal cancer organoids grown in a 3D printed scaffold.

[0098] Example 3: A method for preparing a cell-laden porous 3D scaffold and its application. In this example, it is used to culture colorectal cancer organoids. It adopts an extrusion direct writing printing method, and the steps are as follows:

[0099] B 1. Design of porous 3D bracket model: Based on CAD software, design a cylindrical solid model with a diameter of 12mm and a thickness of 2.5mm. Import the model into the software supporting the printer, set the diameter of the print head and the printing path to be extruded, so that the cylinder can achieve a pore size of 1.2-1.5mm and a wall thickness of 0.4-0.8mm (diameter of the printing filament). After setting, generate a printable executable file (such as Figure 3 shown);

[0100] B2, turn on the extrusion direct writing 3D printer, set the stage temperature to 4°C to pre-cool the printer chamber (this embodiment uses the property of methacrylated gelatin to be in a gel state at low temperatures to achieve thickening), and import the model designed in step B1 into the extrusion direct writing 3D printer;

[0101] B3. Prepare printing ink: prepare RPMI1640 solution of 15% methacryloyl gelatin (GelMA), 1.5% methacryloyl hyaluronic acid (HAMA), 0.04% tartrazine, and 0.75% phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP), and then filter and sterilize with a 0.22 um filter membrane to prepare a mixed solution 1 for mixing with the cell suspension.

[0102] B4. Preparation of colorectal cancer PDO (cell suspension): Take several rice-sized tumor tissues or PDO tissues after passage, wash them several times with pre-cooled phosphate buffered saline (PBS) containing 1× penicillin-streptomycin solution; cut the tissue into minced meat (PDO tissue does not need to be cut); transfer the minced tissue to a centrifuge tube, add an appropriate amount of RPMI 1640 culture medium containing 3 mg / mL of collagenase IV and 5 MU / mL of DNase I, and shake and digest at 37°C for 1 hour; filter the fully lysed cells or cell tissue suspension with 100 μm and 40 μm cell filters in turn to remove residual tissue; centrifuge the filtrate at 1500 rpm for 5 minutes, and remove the supernatant; then use red blood cell lysis buffer to lyse the red blood cells for 5 minutes; the obtained single cell suspension has a cell concentration of 1*10 7 ~1*10 8 , used for subsequent mixing with mixed solution 1;

[0103] B5. Mix the mixed solution 1 and the single-loaded cell suspension in a volume ratio of 2:1, add the mixture to the extrusion printing tube after mixing, and place it in a 4°C refrigerator for 15 minutes to make the solution semi-gel-like; then take out the printing tube and load it on the extrusion direct writing 3D printer, and extrude the porous structure in the wells of the 12-well plate after precooling at 4°C according to the settings of the extrusion direct writing 3D printer, and then use 405nm light for 5s-30s to secondary solidify the gel to achieve the printing of the 3D scaffold;

[0104] B6. After the 3D bracket is printed (the printed 3D bracket is consistent with the designed model, such as Figure 3 The 12-well plate on the stage was taken out, washed with PBS, and then induction medium was added to culture colorectal cancer organoids.

[0105] Example 4: Application of a cell-laden porous 3D scaffold. In this example, it is used for drug toxicity testing of liver organoids, and the steps are as follows:

[0106] C 1. Design a 3D bracket model with multiple holes and inlet 1 and outlet 2 structures, such as Figure 4 As shown in the figure, a porous cylindrical model with closed sides is designed based on CAD software, with a pore size of 1.2-1.5mm and a wall thickness of 0.4-0.6mm; multi-parameter slicing is performed using the supporting software of the 3D printer, and the printing parameters of the bottom layer, chamber layer, and top layer are set respectively, and the model is generated into a printable executable file for subsequent printing on the printer;

[0107] C2. Turn on the light-curing 3D printer, set the temperature to 37°C to preheat the printer chamber, and import the model designed in step C1 into the light-curing 3D printer;

[0108] C3. Prepare non-cell-carrying printing ink: prepare PBS solution (PBS provides appropriate osmotic pressure) of 10% methacryloyl gelatin (GelMA), 10% methacryloyl dextran (DexMA), 0.025% tartrazine, and 0.5% phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP), and then filter and sterilize with a 0.22um filter membrane to prepare a top layer liquid for printing the top layer of the 3D scaffold. Prepare 15% methacryloyl gelatin (GelMA), 1.5% methacryloyl hyaluronic acid (HAMA), 0.03% tartrazine + 0.75% phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP) in DMEM solution as mixed solution 1, so as to be mixed with the cell-carrying suspension for printing of cell-carrying porous 3D scaffolds;

[0109] C4. Preparation of cell-carrying printing ink: Take HepaRG cells that have passed the quality inspection and resuspend them in DMEM solution containing 10% fetal bovine serum (FBS), 1× double antibody, 2mM glutamine, 5ug / ml insulin, 0.5mM hydrocortisone, and 1.25mM acetylcysteine ​​(for subsequent culture medium). The cell concentration is 1*10 6 ~1*10 7 , configured into a single cell-carrying suspension; mixing the mixed solution 1 with the single cell-carrying suspension in a volume ratio of 2:1 to configure a cell-carrying printing ink;

[0110] C5. Printing 3D scaffolds with inlets and outlets: Add the cell-carrying printing ink to the preheated liquid tank of the light-curing 3D printer, print the bottom layer of the cell-carrying cavity and the chamber layer (including the porous structure), pause after the chamber layer is printed, replace it with the liquid tank containing the top layer liquid, continue to print the top layer (including the inlet and outlet), and complete the printing of the 3D scaffold (the printed 3D scaffold is consistent with the designed model, such as Figure 4 shown);

[0111] C6. After printing, remove the printed material, wash it with PBS three times, connect the hose to the printed material, rinse the chamber with PBS, and then pass the culture medium into the chamber after rinsing. After culturing for at least 3 days, observe the formation of liver organoids, and use them for subsequent toxicity testing after the state is stable;

[0112] C7. Drug toxicity test: Replace the reservoir with culture medium containing the target drug concentration and culture at a drip rate of 5 ml / 24 h for 3-7 days. Perform cell activity, cell apoptosis, ALT, AST biochemical tests, organoid fatty degeneration, and cholestasis-related index tests in the experimental group, control group, and blank group to analyze drug hepatotoxicity.

[0113] Therefore, in the cell-loaded porous 3D scaffold prepared in the present invention, the cells are located in the porous 3D scaffold, and the cells can fully contact with the external culture medium, test objects, and screening objects, thereby realizing the dynamic and static culture, testing and screening functions of the cells, thereby improving scientific research efficiency.

[0114] In the description of the present invention, it should be understood that the terms "upper", "lower", "top", "bottom", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In the description of the present invention, "plurality" means two or more, unless otherwise clearly and specifically defined.

[0115] In the present invention, unless otherwise clearly specified and limited, the terms "install", "connect", "connect", "fix" and the like should be understood in a broad sense, for example, it can be a fixed connection, or a detachable connection, or an integral one; it can be a mechanical connection, or an electrical connection; it can be directly connected, or indirectly connected through an intermediate medium, for example, the two can form a mechanical abutment or abutment connection through abutment, contact, etc., the two can also be directly hung or hung through an intermediate medium, etc., or it can be the internal connection of the two elements or the interaction relationship between the two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

Claims

1. A method for preparing a cell-laden porous 3D scaffold, comprising the following steps: S1. preparing printing ink; S2, adding the printing ink prepared in step S1 into a 3D printing device, and printing a 3D bracket according to the designed model; The printing ink comprises a main material, an auxiliary material and a cell suspension.

2. The method for preparing a cell-laden porous 3D scaffold according to claim 1, characterized in that: The main material includes one or more mixtures of photosensitive materials and non-photosensitive materials; The photosensitive material comprises one or more of methacrylic collagen, methacrylic gelatin, methacrylic hyaluronic acid, methacrylic silk protein, methacrylic elastin, methacrylic heparin, methacrylic keratin, methacrylic sericin, methacrylic polyvinyl alcohol, methacrylic polycaprolactone, methacrylic chitosan, methacrylic dextran, methacrylic sodium alginate, methacrylic chondroitin sulfate, polyether F127 diacrylate, and polyethylene glycol diacrylate; The non-photosensitive material includes one or more of collagen, gelatin, silk fibroin, keratin, elastin, sodium alginate, chitosan, agarose, polyvinyl alcohol, polycaprolactone, polyurethane, polyethylene glycol, hydroxyapatite, tricalcium phosphate, polyether F127, porcine, bovine, mouse or human decellularized matrix, and nanoclay; The cell suspension contains cells and a culture medium solution suitable for the cells; And / or, the cells in the cell suspension include one or more of a tumor organoid cell suspension extracted from cancerous tissue after subculture, tumor cell line cells, extracted tumor fibroblasts, extracted tumor stem cells, immune cells, and vascular endothelial cells; And / or, the cells contained in the cell suspension also include one or more of organoid cell suspensions extracted from normal tissues and cultured after subculture, tissue cell lines, stem cells extracted from tissues, iPSCs and functional cells differentiated from iPSCs, embryonic stem cells and functional cells differentiated from them, immune cells, and vascular endothelial cells.

3. The method for preparing a cell-laden porous 3D scaffold according to claim 2, characterized in that: The mass ratio of the photosensitive material in the printing ink is 0.1% to 90%, the mass ratio of the non-photosensitive material in the printing ink is 0.1% to 80%; the mass ratio of the auxiliary material in the printing ink is 0% to 50%; the cell content in the cell suspension is 1*10 3 ~1*10 9 .

4. The method for preparing a cell-laden porous 3D scaffold according to claim 1, characterized in that: The 3D printing device is a light-curing 3D printer or an extrusion direct writing 3D printer; When the 3D printing device adopts a light-curing 3D printer, the auxiliary materials include a photoinitiator and a light absorber; When the 3D printing device adopts an extrusion direct writing 3D printer, the auxiliary material includes a thickener; And / or, the photoinitiator includes one or more of Irgacure 2959, phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP), lithium phosphate, TPO, proanthocyanidin, camphorquinone, eosin Y, terpyridine ruthenium chloride and sodium persulfate, dihydro-4,4-dimethyl-2,3-furandione, ethyl pyruvate, and lipoic acid; the light absorber includes one or more of tartrazine, rutin, phenol red, eosin, phthalocyanine blue, curcumin, and brilliant blue; And / or, the thickener includes one or more of carboxymethyl cellulose, polyether F127, lithium magnesium silicate, sodium alginate, chitosan, agarose, and methacrylated gelatin.

5. The method for preparing a cell-laden porous 3D scaffold according to claim 1, characterized in that: In the step S2, the designed model is the first model or the second model, the 3D bracket printed according to the first model is a porous 3D bracket, and the 3D bracket printed according to the second model is a porous 3D bracket with an inlet and an outlet; The 3D scaffold printed according to the first model has a plurality of interconnected holes, and the 3D scaffold contains cells, and the cells can grow in the 3D scaffold; After step S2, the 3D scaffold is placed in a culture dish or culture plate, and after adding culture medium, it is placed in a carbon dioxide incubator for culture, and the culture medium is replaced regularly to provide a static culture environment for the 3D scaffold; The 3D scaffold printed according to the second model has a sealing structure, an inlet and an outlet connected to the sealing structure, the sealing structure has a plurality of holes connected to each other, the inlet and the outlet are respectively connected to the holes in the sealing structure, and the 3D scaffold contains cells, and the cells can grow in the 3D scaffold; After the step S2, the hoses are connected to the inlet and outlet pre-printed on the 3D scaffold respectively, the pump is connected to the reservoir through the hose, the pump, the hose, the reservoir and the 3D scaffold are placed in a carbon dioxide incubator, and the pump is turned on. The pump pumps the culture medium in the reservoir into the inlet of the 3D scaffold through the hose, and the culture medium flows out from the outlet of the 3D scaffold after passing through the interconnected holes in the 3D scaffold, thereby realizing the dynamic flow of the culture medium in the molded 3D scaffold and providing a dynamic culture environment for the 3D scaffold; And / or, after the cell culture is completed, the culture medium is replaced with a test substance or a screening substance to provide a testing or screening environment for the 3D scaffold.

6. The method for preparing a cell-laden porous 3D scaffold according to claim 5, characterized in that: The 3D bracket has a porous grid structure interconnected with each other, the diameter of the grid is 0.1mm to 5mm, and the wall thickness of the grid is 0.05mm to 3mm.

7. A cell-laden porous 3D scaffold, characterized in that: The cell-laden porous 3D scaffold is prepared by the preparation method of any one of claims 1 to 6.

8. A method for applying a cell-laden porous 3D scaffold, characterized in that: Comprising a 3D scaffold, wherein the 3D scaffold comprises a porous 3D scaffold, or the 3D scaffold is a porous 3D scaffold with an inlet and an outlet, and the 3D scaffold is prepared by the preparation method of the cell-laden porous 3D scaffold according to any one of claims 1 to 6; When the 3D scaffold is a porous 3D scaffold, the 3D scaffold is placed in a culture dish or a culture plate, and then the culture medium, the test object or the screening object is added to the culture dish or the culture plate, and the culture dish or the culture plate is placed in a carbon dioxide incubator to achieve a static culture, test or screening environment for the 3D scaffold; When the 3D scaffold is a 3D porous scaffold with an inlet and an outlet, the hoses are respectively connected to the inlet and outlet pre-printed on the 3D scaffold, the pump is connected to the reservoir through the hose, culture medium, test material or screening material is added to the reservoir, the pump, hose, reservoir and 3D scaffold are placed in a carbon dioxide incubator and the pump is turned on. The pump pumps the culture medium, test material or screening material in the reservoir into the inlet of the 3D scaffold through the hose, and the culture medium, test material or screening material flows out from the outlet of the 3D scaffold after passing through the interconnected pores in the 3D scaffold, thereby realizing dynamic culture, dynamic testing or dynamic screening of the 3D scaffold.

9. The method for applying the cell-laden porous 3D scaffold according to claim 8, characterized in that: The test substance is one or more of small molecule chemical drugs, albumin conjugates and liposomes of small molecule chemical drugs, radioactive small molecule chemical drugs, macromolecular protein drugs, nucleic acid drugs, nanoparticle drugs or cell therapy drugs.

10. The method for applying the cell-laden porous 3D scaffold according to claim 8, characterized in that: The screening object is one or more of peripheral blood mononuclear cells, universal T cells, NK cells, macrophages differentiated from iPSCs, stem cells extracted from tissues, tumor-infiltrating T cells, and CAR-modified T cells or NK cells.

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