Preparation method and application of multi-material cell-loaded 3D scaffold

Abstract

The invention discloses a preparation method and application of a multi-material cell-loaded 3D scaffold, and the preparation method comprises the following steps: S1, preparing at least two kinds of printing ink; s2, various printing inks prepared in the step S1 are added into 3D printing equipment, and a 3D support is printed according to a designed model; the printing ink comprises a main material and a photoinitiator, the main material comprises one or a mixture of a photosensitive material and a non-photosensitive material, and at least one kind of printing ink further comprises a cell-loaded suspension. The processing and manufacturing efficiency and convenience can be improved, the manufacturing cost is reduced, meanwhile, dynamic culture and testing of the carrier cells are achieved, 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 and application of a multi-material cell-laden 3D scaffold. Background Art

[0002] At present, the mainstream organoid culture in the world is mainly divided into two main culture methods: conventional culture plate culture and high-throughput microfluidics (i.e., organoid chip, Organ-on-chip, OOC). Conventional culture plate culture generally uses matrix gel and special culture medium containing growth factors to culture cells statically. This culture method requires manual regular replacement of culture medium and cannot achieve dynamic culture.

[0003] As a rapidly developing science and technology, microfluidic chip technology has demonstrated its unique advantages in the field of biomedicine and has become an innovative method for organoid culture. Organoid chips generally colonize cells in a chamber in the middle of a microfluidic flow channel, and pump the culture medium containing substances required for organoid growth and differentiation into the cell growth chamber in the chip through a single or several inlets and outlets through a peristaltic pump, realizing automatic culture medium replacement, and improving throughput and culture efficiency by connecting multiple microchannels and chambers in series and parallel.

[0004] Organoid chips are generally made of PDMS and PMMA materials through photoetching or machining. After preparation, they need to be sterilized and loaded with corresponding cells. At the same time, the chamber must be sealed and connected to a peristaltic pump for use. This technology achieves high throughput and improves culture efficiency. It can be used in dynamic cell culture, drug sensitivity and drug screening. However, the technical threshold of organoid chips is high, and different chips need to be customized for different studies. The production cost is high and the cycle is long, which limits its application. Therefore, how to solve the above technical problems is the direction that technicians in this field need to work hard. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method and application of a multi-material cell-loaded 3D scaffold. By using this method and application, it is possible to improve production efficiency and convenience and reduce production costs. At the same time, it is also possible to provide a multi-material cell-loaded complex structure for multi-cell co-culture, simulate the microenvironment of cell growth to the greatest extent, realize dynamic culture and testing functions of cells, and improve scientific research efficiency.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a method for preparing a multi-material cell-laden 3D scaffold, the steps of which are:

[0007] S1. preparing printing inks, wherein the printing inks are at least two types;

[0008] S2, adding the various printing inks prepared in step S1 into a 3D printing device, and printing a 3D bracket according to the designed model;

[0009] The printing ink comprises a main material and a photoinitiator, wherein the main material comprises one or more mixtures of photosensitive materials and non-photosensitive materials, and at least one printing ink further comprises a cell-carrying suspension.

[0010] In the above technical solution, the 3D bracket has an inlet and an outlet, and the 3D bracket has a microchannel connected to the inlet and the outlet. After step S2, the 3D bracket is placed in a culture dish, the culture tube is connected to the pre-printed inlet and outlet, the peristaltic pump, the syringe pump or the air pump is connected to the liquid reservoir through the culture tube, and the peristaltic pump, the syringe pump or the air pump are placed in a carbon dioxide incubator and then turned on. The peristaltic pump, the syringe pump or the air pump pumps the culture medium in the liquid reservoir into the inlet of the 3D bracket through the culture tube, and the culture medium flows out from the outlet of the 3D bracket after passing through the microchannel in the 3D bracket, thereby realizing the circulation of the culture medium in the formed 3D bracket and providing a dynamic culture environment for the 3D bracket.

[0011] And / or, the outer side of the 3D scaffold has at least one liquid storage tank, and the inside of the 3D scaffold has a microchannel connected to the liquid storage tank. After step S2, the 3D scaffold is placed in a culture dish, the culture medium is poured into the liquid storage tank, the culture dish carrying the 3D scaffold is placed on a shaker, and the shaker is placed in a carbon dioxide incubator, the shaker is turned on and moved, and the culture medium in the liquid storage tank is allowed to circulate or reciprocate through the microchannel due to the action of gravity or centrifugal force, so as to realize the circulation of the culture medium in the formed 3D scaffold and provide a dynamic culture environment for the 3D scaffold;

[0012] And / or, the outer end of the 3D scaffold has at least two struts, and the struts are connected to the stretching device. After step S2, the 3D scaffold is placed in a culture dish, and the culture medium is poured into the culture dish. The culture dish carrying the 3D scaffold and the stretching device connected to the struts are placed in a carbon dioxide incubator together, and the stretching device is turned on to move, so that the 3D scaffold reciprocates under the stretching force, providing a mechanical dynamic culture environment for the 3D scaffold. Among them, the 3D scaffold with struts can also be printed with a liquid storage tank, and while being stretched by the stretching device and the struts, it can also be placed in a shaking table to cooperate to achieve dynamic culture.

[0013] Among them, the 3D bracket can have an inlet, an outlet, a liquid storage tank and a support rod at the same time, or it can have an inlet, an outlet and a liquid storage tank, or a liquid storage tank and a support rod, or an inlet, an outlet and a support rod. It can also be used in conjunction with various combinations of peristaltic pumps / syringe pumps / air pumps, shakers, and stretching devices, with stronger functions and greater applicability.

[0014] In the above technical solution, after the culture of the carrier cells is completed or the 3D scaffold is directly printed using the cultured carrier cells, the culture medium is replaced with the test object, and the above steps are repeated to provide a dynamic test environment for the 3D scaffold.

[0015] 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%, and the mass ratio of the photoinitiator in the printing ink is 0.1% to 10%;

[0016] The photosensitive material further comprises an aqueous solution or an organic solution, and the aqueous solution or the organic solution accounts for 1% to 99% of the mass ratio of the photosensitive material.

[0017] In the above technical solution, the photosensitive material includes one or more of methacrylic acid-treated collagen, methacrylic acid-treated gelatin, methacrylic acid-treated hyaluronic acid, methacrylic acid-treated silk fibroin, methacrylic acid-treated elastin, methacrylic acid-treated heparin, methacrylic acid-treated keratin, methacrylic acid-treated sericin, methacrylic acid-treated polyvinyl alcohol, methacrylic acid-treated polycaprolactone, methacrylic acid-treated chitosan, methacrylic acid-treated dextran, methacrylic acid-treated sodium alginate, methacrylic acid-treated chondroitin sulfate, polyether F127 diacrylate, and polyethylene glycol diacrylate;

[0018] The non-photosensitive material includes one or more of collagen, gelatin, silk fibroin, keratin, elastin, fibronectin, agarose, sodium alginate, polyvinyl alcohol, polycaprolactone, polyurethane, polyethylene glycol, hydroxyapatite, tricalcium phosphate, polyether F127, porcine, bovine, mouse or human decellularized matrix, and nanoclay;

[0019] The photoinitiator includes one or more of Irgacure 2959, phenyl-2,4,6-trimethylbenzoyl lithium phosphite, 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;

[0020] And / or, the cells in the cell suspension include one or more of fibroblasts, vascular endothelial cells, epithelial keratinocytes, nerve cells, muscle cells, chondrocytes, osteoblasts, intestinal epithelial cells, hepatocytes, lung epithelial cells, immune cells, muscle stem cells, embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, epidermal stem cells, adipose stem cells, stem cells and functional cells differentiated from the above cells, organoid suspensions extracted from various normal or cancerous tissues and completed after subculture, and exosome vesicles secreted by cells.

[0021] In the above technical solution, in step S2, the designed model includes multiple structures, and the multiple structures are combined into the model;

[0022] Each of the structures is printed using a printing ink.

[0023] In the above technical solution, the 3D printing device has a material tank, and before the 3D bracket is printed, the printing ink is placed in the material tank;

[0024] The number of the material trough is one or more;

[0025] When there is only one material tank, after one structure is printed, the printing device is paused, the printing ink in the material tank is replaced with the printing ink required for another structure, and then the printing device is canceled and the structure printing is continued;

[0026] When there are multiple material slots, each type of printing ink is placed in a corresponding material slot. During the process of printing the 3D bracket by the 3D printing device, the printing ink in the corresponding material slot is automatically selected or switched to print the 3D bracket.

[0027] In the above technical solution, during the 3D bracket printing process, when it is necessary to replace different printing inks to print the 3D bracket, negative pressure suction is used in advance and / or a motor is installed on the printing platform to rotate and dry the residual printing ink in the 3D bracket that has been printed on the printing platform to clean it, and then other printing inks are used to continue printing the 3D bracket.

[0028] The present invention also provides a multi-material cell-laden 3D scaffold, which is prepared by the above-mentioned preparation method of the multi-material cell-laden 3D scaffold.

[0029] The printing ink used in the inlet and outlet is the same as or different from the printing ink of the main body;

[0030] The cell-carrying 3D scaffold comprises a main body having cells and microchannels;

[0031] The main body is provided with an inlet and an outlet, and two ends of the microchannel are respectively connected with the inlet and the outlet;

[0032] And / or, the main body has at least one liquid storage groove, and both ends of the microchannel are respectively connected to one or more of the liquid storage grooves;

[0033] And / or, the main body has at least two support rods, and the support rods are connected to the stretching device.

[0034] The present invention also provides an application of a multi-material cell-carrying 3D scaffold, comprising a 3D scaffold, wherein the 3D scaffold is placed in a culture dish, a culture medium is added to a storage tank of the culture dish or the 3D scaffold, and the culture dish is placed in a carbon dioxide incubator to provide a dynamic culture or test environment for the 3D scaffold;

[0035] The 3D scaffold is prepared by the above-mentioned method for preparing the multi-material cell-laden 3D scaffold.

[0036] In the above technical solution, the 3D bracket has an inlet and an outlet, and the 3D bracket has a microchannel connected to the inlet and the outlet. The 3D bracket is placed in a culture dish, and the culture tube is connected to the pre-printed inlet and outlet. The peristaltic pump, the syringe pump or the air pump is connected to the liquid reservoir through the culture tube. After the 3D bracket is placed in a carbon dioxide incubator, the peristaltic pump, the syringe pump or the air pump is turned on. The peristaltic pump, the syringe pump or the air pump pumps the culture medium in the liquid reservoir into the inlet of the 3D bracket through the culture tube, and the culture medium flows out from the outlet of the 3D bracket after passing through the microchannel in the 3D bracket, thereby realizing the circulation of the culture medium in the molded 3D bracket and providing a dynamic culture environment for the 3D bracket.

[0037] And / or, the outer side of the 3D scaffold has at least one liquid storage tank, and the inside of the 3D scaffold has a microchannel connected to the liquid storage tank. After step S2, the 3D scaffold is placed in a culture dish, the culture medium is poured into the liquid storage tank, the culture dish carrying the 3D scaffold is placed on a shaker, and the shaker is placed in a carbon dioxide incubator. The shaker is turned on and moved, and the culture medium in the liquid storage tank circulates or reciprocates through the microchannel due to the action of gravity or centrifugal force, so that the culture medium circulates in the molded 3D scaffold, providing a dynamic culture environment for the 3D scaffold;

[0038] And / or, the outer end of the 3D scaffold has at least two support rods, and the support rods are connected to the stretching device. After step S2, the 3D scaffold is placed in a culture dish, and then the culture medium is poured into the culture dish. The culture dish carrying the 3D scaffold and the stretching device connected to the support rods are placed in a carbon dioxide incubator together, and the stretching device is turned on and moved, so that the 3D scaffold reciprocates under the stretching force, providing a mechanical dynamic culture environment for the 3D scaffold.

[0039] In the above technical solution, after the culture of the carrier cells is completed or the 3D scaffold is directly printed using the cultured carrier cells, the culture medium is replaced with the test object, and the above steps are repeated to provide a dynamic test environment for the 3D scaffold.

[0040] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0041] 1. The present invention directly uses 3D printing to print out a 3D scaffold with cells mixed inside, which can flexibly realize a multi-material complex structure for multi-cell co-culture, and imitate the microenvironment suitable for the growth of different cells to the maximum extent. At the same time, various complex series-parallel microchannel structures are constructed in its structure, thereby realizing dynamic cell culture and testing functions such as drug screening and drug sensitivity detection, effectively improving scientific research efficiency;

[0042] 2. The present invention uses 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 scaffolds with more complex structures, and can also improve the scalability of scientific research;

[0043] 3. In the present invention, before 3D scaffold printing, the cell suspension containing the corresponding culture is directly mixed into the printing ink for 3D printing so that the cells can be evenly distributed in the 3D scaffold, which can improve the culture effect and convenience of the cells;

[0044] 4. The 3D scaffold in the present invention can be printed with the same material or a mixture of different materials, and can be adjusted according to the types of different cells. Using different materials for different cells can provide a more suitable culture environment for specific cells (such as using elastic materials for muscle cells and hard materials for bone cells is more conducive to their growth), thereby optimizing the culture effect;

[0045] 5. The photocuring 3D printing method of the present invention has high precision, and can achieve an optical precision of up to 1 micron, so that complex structures such as micro-pipes can be printed, and complex structures can be realized in a very small area, reducing the number of cells used to achieve the same scientific research purpose, and has the advantages of low cost and high precision;

[0046] 6. The present invention adopts the method of cell-carrying 3D printing to form the main body and microfluidic channel of the organoid chip in one piece at one time. The structure realized is not only similar to the conventional organoid chip, but also can prepare structures such as liquid storage tanks or support rods that conventional organoid chips do not have, thereby constructing a wider range of dynamic culture and testing models with better scalability; this technology has the advantages of short production cycle, low cost and easy operation. For scientific researchers, this technology has great flexibility. Once they have an idea, they can immediately use 3D printing to realize the organoid chip and verify it immediately. They can also realize the construction of a multi-cell co-culture model through multi-structure and multi-material printing, thereby improving their scientific research efficiency and accelerating the research process of regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is a schematic structural diagram of a 3D bracket in Embodiment 1 of the present invention;

[0048] Figure 2: is a schematic diagram of the structure of the 3D stent in Example 2 of the present invention (the inlet and outlet are not shown, and it is a vascularized channel structure carrying vascular endothelial cells);

[0049] Figure 3 is a schematic diagram of the structure of the 3D bracket in the second embodiment of the present invention;

[0050] Figure 4 is a schematic diagram of the structure of the 3D bracket in the third embodiment of the present invention;

[0051] Figure 5 It is a schematic diagram of the structure of the 3D scaffold in the fourth embodiment of the present invention during the cell loading test.

[0052] Among them: 1. main body; 2. microchannel; 3. inlet; 4. outlet; 5. liquid storage tank; 6. support rod; 7. peristaltic pump; 8. liquid storage tank; 9. silicone tube; 10. micro patch. DETAILED DESCRIPTION

[0053] The present invention will be further described below in conjunction with the accompanying drawings and embodiments:

[0054] Example 1: See Figure 1 As shown, a method for preparing a multi-material cell-laden 3D scaffold comprises the following steps:

[0055] S1. preparing printing inks, wherein the printing inks are at least two types;

[0056] S2, adding the various printing inks prepared in step S1 into a 3D printing device, and printing a 3D bracket according to the designed model;

[0057] The printing ink comprises a main material and a photoinitiator, wherein the main material comprises one or more mixtures of photosensitive materials and non-photosensitive materials, and at least one printing ink further comprises a cell-carrying suspension.

[0058] In this embodiment, a 3D scaffold is directly printed out by 3D printing using a variety of printing inks, wherein, since at least one printing ink contains a cell suspension, and the cell suspension contains cells, in this method, the cells are directly mixed into the 3D scaffold, and before the 3D scaffold is printed, the cell suspension selects the cells to be cultured, and in the process of model design of the 3D scaffold, the scaffold is designed according to the most suitable culture space for the cells to be cultured (such as bone cells are printed using a porous scaffold structure that imitates cancellous bone) and the corresponding materials required to simulate the in vivo microenvironment, and then the 3D scaffold is printed, and then the corresponding culture medium is used to culture the cells inside the 3D scaffold or test the test object (the test object is an object that needs to contact with the cells to test its interaction, and the test object is an aqueous solution of drugs, immune cells, etc.), so that the in vivo microenvironment can be simulated to the greatest extent. At the same time, a variety of cells can be pre-printed in the 3D scaffold and the corresponding cell structure can be designed, so that a multi-material complex structure for multi-cell co-culture can be flexibly realized, the production cost is low, and the dynamic culture and testing of cells can be realized, which effectively improves the efficiency of scientific research.

[0059] The present invention also provides a cell-laden 3D scaffold, which is prepared by the above-mentioned preparation method of the multi-material cell-laden 3D scaffold.

[0060] The cell-carrying 3D scaffold comprises a main body 1 having cells and microchannels 2 .

[0061] There are two main ways to dynamically culture or test cells: the first is based on a shaker, and the second is based on microfluidics. The two culture or test methods require different structures for the 3D scaffold.

[0062] Therefore, based on the dynamic culture method of microfluidics (in this embodiment, the culture method is taken as an example), the structure of the 3D scaffold is as follows: the main body has an inlet 3 and an outlet 4, and the two ends of the microchannel are connected to the inlet and outlet respectively, and the cells are directly mixed into the main body. The printing ink used in the inlet and outlet is the same as or different from the printing ink of the main body;

[0063] Wherein, the dynamic culture step based on microfluidics is: the 3D scaffold is placed in a culture dish, the culture tube is connected to the pre-printed import and export, the culture tube is connected to the peristaltic pump (syringe pump or air pump, one of the three is selected, in the present embodiment, the peristaltic pump is used as an example) and the reservoir (the reservoir is outside), and the peristaltic pump, syringe pump or air pump are turned on after being placed in a carbon dioxide incubator. The peristaltic pump, syringe pump or air pump pumps the culture medium in the reservoir into the 3D scaffold import through the culture tube, and flows out from the 3D scaffold outlet after passing through the microchannel in the 3D scaffold, so as to realize the circulation of the culture medium in the molded 3D scaffold, and provide a dynamic culture environment of the 3D scaffold. Wherein, the microchannel is a microstructure (bionic vascular structure) that conforms to the corresponding cell-carrying culture. In this process, when the culture medium flows through the channel, it can provide nutrition to the cells in the 3D scaffold, so as to realize the circulation of the culture medium in the molded 3D scaffold, and provide a dynamic culture environment of the vascularized class of the cell-carrying scaffold. At the same time, the carrier cells are tested with the test material. After the carrier cells are cultured, the test material is replaced with the culture medium, and the above steps are repeated. In the present invention, in other embodiments, or in other culture methods, the test method is the same. After the carrier cells are cultured or the cultured carrier cells are directly used to print the 3D scaffold, the culture medium is replaced with the test material for testing. During the test, the test material is used to contact the carrier cells, and then the interaction between the carrier cells is tested. In this way, the 3D scaffold can culture and test the carrier cells.

[0064] Among them, in this embodiment, since the inlet and outlet need to be connected to the culture tube, they require a certain hardness and strength, and also a certain supporting capacity. Moreover, there is no need to culture cells there. Therefore, in this embodiment, the printing ink at the inlet and outlet of the main body may not contain the cell suspension, which can increase the strength of the inlet and outlet (the mechanical properties of the cell-carrying material are insufficient, therefore, the printing ink at the inlet and outlet does not contain the cell-carrying material, has better strength and stronger mechanical properties, thereby facilitating connection with the culture tube), and can also save the material for the cell-carrying material and reduce the production cost.

[0065] Based on the dynamic culture method of the shaking table, the structure of the 3D scaffold is as follows: the main body 1 is provided with two liquid storage tanks 5, and the two ends of the microchannel are respectively connected to the two liquid storage tanks.

[0066] Among them, the dynamic culture steps based on the shaker are: put the 3D scaffold into the culture dish, pour the culture medium into the left and right liquid storage tanks, put the culture dish containing the 3D scaffold on the shaker, and put the shaker into the carbon dioxide incubator together, turn on the shaker to move, and swing the shaker up and down to allow the culture medium in the liquid storage tank to circulate through the microchannel under the action of gravity, so as to realize the reciprocating flow of the culture medium in the molded 3D scaffold and provide a dynamic culture environment for the 3D scaffold. In this way, the microchannel is a microstructure that conforms to the corresponding cell culture, and the liquid storage tank is mainly used for the storage of the culture medium and does not need to be used for the culture of cells. Therefore, the printing ink used here may not contain cells, so that the strength and support performance can be better and the cost can be lower. (If it is a test, the culture medium is replaced with the test object)

[0067] Of course, in this embodiment, two liquid storage tanks are provided on the main body. In other embodiments, only one liquid storage tank may be provided on the main body, and the liquid storage tank is connected to the microchannel. In this way, the culture medium in the liquid storage tank flows into the microchannel through the swing of the shaking table, and then flows back to the liquid storage tank from the microchannel, which can also realize the circulation of the culture medium in the microchannel and realize the dynamic culture of the cells.

[0068] Among them, since the printing ink contains the main material and the photoinitiator, and the presence of the photoinitiator is mainly to solidify the material of the structure part of the light-irradiated part through the effect of light excitation during the 3D printing process, there may be residual photoinitiator, which will affect the normal culture of the cells. Therefore, after printing the 3D scaffold in step S2, rinse with PBS buffer for 3 to 5 times to remove the residual photoinitiator in the printing ink.

[0069] Furthermore, in order to ensure the strength of the 3D scaffold, a secondary light curing is performed by light irradiation before rinsing with PBS buffer, thereby strengthening the strength of the 3D scaffold. Of course, since light irradiation will affect the activity of the cells, this step is not a necessary step.

[0070] After the 3D scaffold is rinsed with PBS buffer, the cells are cultured with culture medium (dynamic culture based on a shaker or dynamic test based on a shaker).

[0071] 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%, and the mass ratio of the photoinitiator in the printing ink is 0.1% to 10%.

[0072] The photosensitive material further comprises an aqueous solution or an organic solution, and the aqueous solution or the organic solution accounts for 1% to 99% of the mass ratio of the photosensitive material.

[0073] Among them, 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 F127 diacrylate, and polyethylene glycol diacrylate.

[0074] Among them, photosensitive materials are the main materials for photocuring printing. They have the property of quickly changing from liquid to solid under light excitation within a few seconds. They are solidified layer by layer after the 3D printer transmits light (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.

[0075] 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.

[0076] The non-photosensitive material includes one or more of collagen, gelatin, silk fibroin, keratin, elastin, fibronectin, agarose, sodium alginate, polyvinyl alcohol, polycaprolactone, polyurethane, polyethylene glycol, hydroxyapatite, tricalcium phosphate, polyether F127, decellularized matrix of pig, cow, mouse or human origin, and nanoclay.

[0077] Among them, non-photosensitive materials are mainly used to provide the growth microenvironment required by different cells, 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.

[0078] Wherein, the photoinitiator includes one or more of Irgacure 2959, phenyl-2,4,6-trimethylbenzoyl lithium phosphite, 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.

[0079] The cells in the cell suspension include fibroblasts, vascular endothelial cells, epithelial keratinocytes, nerve cells, muscle cells, chondrocytes, osteoblasts, intestinal epithelial cells, hepatocytes, lung epithelial cells, immune cells, muscle stem cells, embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, epidermal stem cells, adipose stem cells, stem cells and functional cells differentiated from the above cells, organoid suspensions extracted from various normal or cancerous tissues after passage culture, and one or more exosome vesicles secreted by cells. The cells in the cell suspension are the cells to be cultured. According to the cells to be cultured, the cell suspension corresponding to the cells to be cultured is mixed into the printing ink, and then the corresponding 3D scaffold is printed according to the cells to be cultured for the culture of the cells.

[0080] Wherein, in the process of printing the 3D bracket, in the step S2, the designed model includes multiple structures, and the multiple structures are combined into the model;

[0081] Each of the structures is printed with a printing ink. At least one of the printing inks contains a cell suspension. In this way, the 3D scaffold after printing has cells inside, and the 3D scaffold can be directly printed into a microscopic and dynamic culture structure that is most suitable for the corresponding cell culture, thereby facilitating scientific research activities such as cell culture and testing.

[0082] The 3D printing device has a material tank, and the printing ink is placed in the material tank before the 3D bracket is printed. The function of the material tank is mainly to place the printing ink and provide printing materials to the 3D printing device.

[0083] Wherein, the material trough is one or more,

[0084] When there is only one material tank, after one structure is printed, the printing device is paused, and after the printing ink in the material tank is cleaned, the printing ink required for another structure is replaced, and then the pause of the printing device is canceled and the structure printing is continued;

[0085] When there are multiple material slots, each type of printing ink is placed in a corresponding material slot. During the process of printing the 3D bracket by the 3D printing device, the printing ink in the corresponding material slot is automatically selected or switched to print the 3D bracket.

[0086] During the 3D bracket printing process, when different printing inks need to be replaced for printing the 3D bracket, negative pressure suction is used in advance or a rotating motor is installed on the printing platform to drive the printing platform to rotate and dry to clean the residual printing ink in the printed 3D bracket, and then other printing inks are used to continue printing the 3D bracket.

[0087] In the present invention, the 3D printer has a variety of structures. In one embodiment (this embodiment), a single cell (a type of carrier cell) multi-material printing method is adopted, and the steps are as follows:

[0088] S1, prepare a printing ink 1 with a mass ratio of 8% methacryloyl gelatin (GelMA) and 15% methacryloyl silk protein (SilMA), add 0.5% of the photoinitiator LAP and mix evenly with the lung epithelial cells (cell suspension) loaded with the culture medium (the rest is an aqueous solution, the content of the aqueous solution is more than 60% of the mass ratio in the corresponding printing ink, the aqueous solution is water, culture medium or pbs solution, and most of the pbs and culture medium are also water), put the prepared printing ink 1 into the material tank of the 3D printer, turn on the 3D printer, and the 3D printer prints the printing ink 1 layer by layer according to the pre-designed structure 1 with a pipeline structure;

[0089] S2, (based on a single-material light-curing 3D printer without automation function): After the structure with microchannels is printed, the 3D printer is paused, the material tank is taken out from the 3D printer, and a material tank containing printing ink 2 is placed (printing ink 2 is 5% methacrylated gelatin GelMA and 20% polyethylene glycol diacrylate (PEGDA) with a mass ratio of 0.3% photoinitiator (LAP) and deionized water mixed evenly) and then printing is continued to print the inlet and outlet structures of the non-cell-bearing body (the inlet and outlet structures are structure 2, which is convenient for inserting a capillary silicone culture tube for internal and external circulation of the culture medium) to complete the printing of the 3D scaffold;

[0090] S3: After printing, remove the 3D scaffold and rinse it with PB S for 3-5 times to remove the residual photoinitiator in the printing ink;

[0091] S4 (dynamic culture based on microfluidics): Place the 3D scaffold in a culture dish, connect the capillary silicone tube to the non-cell-carrying main inlet and outlet of structure 2 respectively, connect the silicone tube to the peristaltic pump (or air pump, or injection pump, taking the peristaltic pump as an example) and the liquid storage tank, put them together in a carbon dioxide incubator and turn on the peristaltic pump. The peristaltic pump pumps the culture medium into the inlet of the 3D scaffold through the culture tube, and then flows out from the outlet of the 3D scaffold after passing through the microchannel structure in the 3D scaffold, thereby realizing the circulation of the culture medium in the molded composite scaffold and providing a dynamic culture environment for the cell-carrying scaffold.

[0092] The cells in the 3D scaffold are lung epithelial cells, which are used to culture lung epithelial cells. Figure 1 shown.

[0093] Example 2: See Figure 2 , 3As shown, a preparation method and application of a multi-material cell-carrying 3D scaffold, which uses multiple cell-carrying cells and multiple materials for printing, and the steps are:

[0094] S1: Prepare a printing ink 1 with a mass ratio of 3% methacryloyl gelatin (GelMA) and 30% polyethylene glycol diacrylate (PEGDA) (the rest is an aqueous solution, the content of the aqueous solution is more than 60% of the mass ratio in the corresponding printing ink, the aqueous solution is water, culture medium or pbs solution, and most of the pbs and culture medium are also water), add 0.5% of photoinitiator (LAP) and deionized water to mix evenly, put the prepared printing ink 1 into the material tank of the 3D printer, turn on the 3D printer, and the 3D printer performs photocuring stacking printing on the printing ink 1 layer by layer according to the pre-designed structure 1, and pause the printer after the required structure 1 is printed;

[0095] S2: Prepare a printing ink 2 with a mass ratio of 10% methacryloyl gelatin (GelMA) and 2% methacryloyl hyaluronic acid (HAMA) (the rest is an aqueous solution, the content of the aqueous solution is more than 60% of the mass ratio in the corresponding printing ink, the aqueous solution is water, culture medium or pbs solution, and most of the pbs and culture medium are also water), add 0.5% of a photoinitiator (LAP) and mix evenly with a vascular endothelial cell suspension (cell suspension) containing a culture medium, load the prepared printing ink 2 into a new material tank, replace the material tank containing printing ink 1 with the material tank containing printing ink 2, and cancel the pause, the 3D printer performs layer-by-layer photocuring stacking printing on the printing ink 2 according to the pre-designed structure 2, pause the 3D printer after the required structure 2 is printed, and take out the printing platform of the bracket containing the printed structure 2;

[0096] S3: Prepare a printing ink 3 with a mass ratio of 8% methacryloyl gelatin (GelMA) and 5% methacryloyl silk protein (SilMA) (the rest is an aqueous solution, the content of the aqueous solution is more than 60% of the mass ratio in the corresponding printing ink, the aqueous solution is water, culture medium or PB S solution, and most of the body of PB S and culture medium is also water), add 0.5% of photoinitiator (LAP) and mix evenly with the pre-cultured neural functional cells (cell suspension) containing culture medium, pour the prepared printing ink 3 into the cavity of the unfinished printing bracket (in the pipe gap in structure 2), turn on the light source with a wavelength of 405nm and irradiate for 30s-60s until the printing ink 3 solidifies into a gel (the printing ink 3 is mixed in structure 2), and put the printing platform back to the original position of the printer;

[0097] S4: Prepare a printing ink 4 with a mass ratio of 3% methacryloyl gelatin (GelMA) and 30% polyethylene glycol diacrylate (PEGDA) (the rest is an aqueous solution, the content of the aqueous solution is more than 60% of the mass ratio in the corresponding printing ink, the aqueous solution is water, culture medium or pbs solution, and most of the pbs and culture medium are also water), add 0.5% of photoinitiator (LAP) and deionized water to mix evenly, put the prepared printing ink 4 into a new material tank, replace the material tank with the material tank containing the printing ink 4 and cancel the pause, the 3D printer performs layer-by-layer photocuring stacking printing on the printing ink 4 according to the pre-designed structure 3, and after the required structure is printed, a formed 3D bracket is made;

[0098] S5: Remove the 3D scaffold from the printing platform and rinse it with PB S for 3-5 times to remove the residual photoinitiator in the printing ink;

[0099] S6: Place the 3D scaffold in a culture dish, and use capillary silicone tubes to connect the inlet and outlet of the non-cell-carrying pipes of structures 1 and 3 respectively. The silicone tubes are connected to a peristaltic pump (or an air pump, or a syringe pump, taking a peristaltic pump as an example) and a liquid reservoir (the liquid reservoir is a separate structure, which is not arranged on the 3D scaffold and is used to place culture medium and test objects). Put them all in a carbon dioxide incubator and turn on the peristaltic pump. The peristaltic pump pumps the culture medium in the liquid reservoir into the inlet of the 3D scaffold through the culture tube, and then flows out of the 3D scaffold outlet after passing through the 3D direct microchannel structure and then enters the liquid reservoir, thereby realizing the circulation of the culture medium in the molded 3D scaffold and providing a dynamic culture environment for the cell-carrying scaffold.

[0100] The cells in the 3D scaffold are pre-cultured neural functional cells and vascular endothelial cells embedded in the microchannels of the main body. The two cells are combined together for the culture of brain organoids. The 3D scaffold structure is as follows Figure 2 , 3 As shown. The top and bottom are structures without cells, and the four holes on the four sides are winding holes for fixing the silicone tube. Since there are no cells, the strength and support performance are better. Figure 2 The main import and export are not shown. Figure 2 This is structure 2 in this embodiment, Figure 3 In the figure, structure 2 is between structure 1 and structure 3, structure 2 is in the middle, structure 1 is above structure 2, and structure 3 is below structure 2.

[0101] If testing is required, the culture medium in step S6 is replaced with the test material, and step S6 is repeated to provide a dynamic testing environment for the 3D scaffold.

[0102] Example 3: See Figure 4As shown, a preparation method and application of a multi-material cell-carrying 3D scaffold, which uses a single cell-carrying and multiple materials for printing, and the steps are:

[0103] S1: (3D printer based on automatic material changing and cleaning tank) prepare a printing ink 1 with a mass ratio of 3% methacryloyl gelatin (GelMA) and 30% polyethylene glycol diacrylate (PEGDA) (the rest is an aqueous solution, the content of the aqueous solution is more than 60% of the mass ratio in the corresponding printing ink, the aqueous solution is water, culture medium or pbs solution, and most of the pbs and culture medium are also water), add 0.5% of photoinitiator (LAP) and deionized water to mix evenly, put the prepared printing ink into the material tank 1 of the 3D printer, turn on the 3D printer, and the printer performs layer-by-layer photocuring stacking printing on the printing ink 1 according to the pre-designed structure 1. After the required structure 1 is printed, the printer will automatically pause, and the printing platform will be lifted out of the material tank 1 and reach the negative pressure cleaning area. After arriving, the printer turns on the negative pressure, and the printed structure 1 on the printing platform (attached Figure 4 The left side of the structure 1) shows that the residual printing ink 1 is drawn out by negative pressure;

[0104] S2: Prepare a printing ink 2 with a mass ratio of 8% methacryloyl gelatin (GelMA) and 15% methacryloyl silk protein (SilMA) (the rest is an aqueous solution, the content of the aqueous solution is more than 60% of the mass ratio in the corresponding printing ink, the aqueous solution is water, culture medium or pbs solution, and most of the pbs and culture medium are also water), add 0.5% of photoinitiator (LAP) and mix evenly with muscle cells containing culture medium (cell suspension), load the prepared printing ink 2 into the material tank 2, and the 3D printer will lift the printing platform into the material tank 2 after completing the negative pressure cleaning in step S1, and perform layer-by-layer photocuring stacking printing on the printing ink 2 according to the pre-designed structure 2. After the required structure 2 is printed, the 3D printer will automatically pause, and the printing platform will reach the negative pressure cleaning area after being lifted out of the material tank 2. After arriving, the 3D printer turns on the negative pressure, and the printed structure 2 (attached Figure 4 The middle part of the structure is structure 2, and the left side of structure 2 is structure 1) The residual printing ink 2 on the structure is sucked out by negative pressure;

[0105] S3: Prepare a printing ink 3 with a mass ratio of 3% methacryloyl gelatin (GelMA) and 30% polyethylene glycol diacrylate (PEGDA) (the rest is an aqueous solution, the content of the aqueous solution is more than 60% of the mass ratio in the corresponding printing ink, the aqueous solution is water, culture medium or pbs solution, and most of the pbs and culture medium are also water), add 0.5% of photoinitiator (LAP) and deionized water to mix evenly, and put the prepared printing ink 3 into the material tank 3. After the 3D printer completes the negative pressure cleaning in step S2, the printing platform will be lifted into the material tank 3, and the printing ink 3 is photocured and stacked layer by layer according to the pre-designed structure. In the required structure 3 (structure 3 is in the vicinity of Figure 4 The structure 3 is on the right side of the structure 2, and the structures 1 and 3 are symmetrically arranged on the left and right sides of the structure 2 respectively) after the printing is completed, the printing is completed to form a composite 3D bracket;

[0106] S4: remove the formed composite 3D scaffold from the printing platform and rinse it with PB S for 3-5 times to remove the residual photoinitiator in the printing ink;

[0107] S5: Place the formed composite 3D scaffold in a culture dish, pour the culture medium into the liquid storage tank in the 3D scaffold, lay it flat, insert the support rod 6 into the symmetrical structure formed by the printing ink 1 and 3, connect the support rod to the stretching device (the stretching device is used to drive the support rod 6 to move unidirectionally or reciprocatingly), place the culture dish carrying the 3D scaffold, the support rod and the stretching device connected to the support rod on a shaker, and put them together in a carbon dioxide incubator, turn on the stretching device, and drive the support rod 6 to move through the stretching device, so that the 3D scaffold performs a cyclic motion under the action of the stretching force, so that the structure carrying muscle cells in the middle can be stretched and contracted, and observe the relevant research on the effect of applying external force on its cells. At the same time, the shaker moves up and down, and the liquid in the liquid storage tank 5 is reciprocated through the microchannel 2 to provide dynamic culture fluid to the main body 1.

[0108] The 3D support structure can be found in Figure 4 shown.

[0109] If testing is required, the culture medium in step S5 is replaced with the test material, and step S5 is repeated to provide a dynamic testing environment for the 3D scaffold.

[0110] Example 4: See Figure 5 As shown, a preparation method and application of a multi-material cell-carrying 3D scaffold, which uses a single cell-carrying and multiple materials for printing, and the steps are:

[0111] S1: (3D printer based on automatic material changing and cleaning tank) prepare a printing ink 1 with a mass ratio of 2% methacryloyl gelatin (GelMA) and 50% polyethylene glycol diacrylate (PEGDA) (the rest is an aqueous solution, the content of the aqueous solution is more than 40% of the mass ratio in the corresponding printing ink, the aqueous solution is water, culture medium or pbs solution, and most of the pbs and culture medium are also water), add 0.5% of photoinitiator (LAP) and deionized water to mix evenly, put the prepared printing ink into the material tank 1 of the 3D printer, turn on the 3D printer, and the printer performs layer-by-layer photocuring stacking printing on the printing ink 1 according to the pre-designed microfluidic structure 1. After the required structure 1 is printed, the printer will automatically pause, and the printing platform will be lifted out of the material tank 1 and reach the negative pressure cleaning area. After arriving, the printer turns on the negative pressure, and the residual printing ink 1 on the printed structure 1 on the printing platform is extracted by the negative pressure;

[0112] S2: Prepare a printing ink 2 with a mass ratio of 10% methacryloyl gelatin (GelMA) and 1% methacryloyl hyaluronic acid (HAMA) (the rest is an aqueous solution, the content of the aqueous solution is more than 60% of the mass ratio in the corresponding printing ink, the aqueous solution is water, culture medium or pbs solution, and most of the pbs and culture medium are also water), add 0.5% of the photoinitiator (LAP) and mix evenly with the pre-cultured hepatocyte organoid (cell suspension) containing the culture medium, and load the prepared printing ink 2 into the material tank 2. After the negative pressure cleaning in step S1 is completed, the printing platform of the 3D printer will be lifted into the material tank 2, and the printing ink 2 will be photocured and stacked layer by layer according to the pre-designed structure 2. After the required structure 2 is printed, the 3D printer ends printing to form a formed composite 3D scaffold;

[0113] S3: After printing, remove the 3D scaffold and rinse it with PB S for 3-5 times to remove the residual photoinitiator in the printing ink;

[0114] S4 (detection and culture based on microfluidics): Place the 3D scaffold in a culture dish, connect the capillary silicone tube to the non-cell-bearing main inlet and outlet of structure 1 respectively, and connect the silicone tube 9 to the peristaltic pump 7 (or air pump, or injection pump, taking the peristaltic pump as an example) and the liquid reservoir 8. Put them together in a carbon dioxide incubator and turn on the peristaltic pump. The peristaltic pump pumps the culture medium in the liquid reservoir into the inlet of the 3D scaffold through the culture tube, and then flows out from the outlet of the 3D scaffold after passing through the microchannel 2 structure in the 3D scaffold, thereby realizing the circulation of the culture medium in the molded composite scaffold and providing a dynamic culture environment for the 3D scaffold.

[0115] S5: After the cell culture is completed, the culture medium in step S4 is replaced with the test object, and step S4 is repeated to provide a dynamic test environment for the 3D scaffold.

[0116] During the test, the cells in the 3D scaffold are cultured liver organoids, which are used for dynamic contact between the liver organoids and the test substance, and the toxicity of the drug is tested by detecting the biological changes of the liver organoids after contact with the test substance. Figure 5 shown.

[0117] Among them, Figure 5 In the figure, the main body and the microchannel are printed with printing ink 1, and the circle arranged on the microchannel constitutes a micropatch 10, which is printed with printing ink 2. The microchannel allows the culture medium / test material to be tested to flow through the micropatch, and the liver organoids are evenly loaded in the micropatch.

[0118] 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.

[0119] 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 multi-material cell-laden 3D scaffold, comprising the following steps: S1. preparing printing inks, wherein the printing inks are at least two types; S2, adding the various printing inks 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 and a photoinitiator, wherein the main material comprises one or more mixtures of photosensitive materials and non-photosensitive materials, and at least one printing ink further comprises a cell-carrying suspension.

2. The method for preparing a multi-material cell-laden 3D scaffold according to claim 1, characterized in that: The 3D support has an inlet and an outlet, and the 3D support has a microchannel connected to the inlet and the outlet. After step S2, the 3D support is placed in a culture dish, and the culture tube is connected to the pre-printed inlet and outlet. The peristaltic pump, the syringe pump or the air pump is connected to the liquid reservoir through the culture tube. After the 3D support is placed in a carbon dioxide incubator, the peristaltic pump, the syringe pump or the air pump is turned on. The peristaltic pump, the syringe pump or the air pump pumps the culture medium in the liquid reservoir into the inlet of the 3D support through the culture tube, and the culture medium flows out from the outlet of the 3D support after passing through the microchannel in the 3D support, thereby realizing the circulation of the culture medium in the formed 3D support and providing a dynamic culture environment for the 3D support. And / or, the outer side of the 3D scaffold has at least one liquid storage tank, and the inside of the 3D scaffold has a microchannel connected to the liquid storage tank. After step S2, the 3D scaffold is placed in a culture dish, the culture medium is poured into the liquid storage tank, the culture dish carrying the 3D scaffold is placed on a shaker, and the shaker is placed in a carbon dioxide incubator, the shaker is turned on and moved, and the culture medium in the liquid storage tank is allowed to circulate or reciprocate through the microchannel due to the action of gravity or centrifugal force, so as to realize the circulation of the culture medium in the formed 3D scaffold and provide a dynamic culture environment for the 3D scaffold; And / or, the outer end of the 3D scaffold has at least two support rods, and the support rods are connected to the stretching device. After step S2, the 3D scaffold is placed in a culture dish, and then the culture medium is poured into the culture dish. The culture dish carrying the 3D scaffold and the stretching device connected to the support rods are placed in a carbon dioxide incubator together, and the stretching device is turned on and moved, so that the 3D scaffold reciprocates under the stretching force, providing a mechanical dynamic culture environment for the 3D scaffold.

3. The method for preparing a multi-material cell-laden 3D scaffold according to claim 2, characterized in that: After the cell culture is completed or the 3D scaffold is directly printed using the cultured cells, the culture medium is replaced with the test object, and the above steps are repeated to provide a dynamic test environment for the 3D scaffold.

4. The method for preparing a multi-material cell-laden 3D scaffold according to claim 1, 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%, and the mass ratio of the photoinitiator in the printing ink is 0.1% to 10%; The photosensitive material further comprises an aqueous solution or an organic solution, and the aqueous solution or the organic solution accounts for 1% to 99% of the mass ratio of the photosensitive material.

5. The method for preparing a multi-material cell-laden 3D scaffold according to claim 1, characterized in that: 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; The non-photosensitive material includes one or more of collagen, gelatin, silk fibroin, keratin, elastin, fibronectin, agarose, sodium alginate, polyvinyl alcohol, polycaprolactone, polyurethane, polyethylene glycol, hydroxyapatite, tricalcium phosphate, polyether F127, porcine, bovine, mouse or human decellularized matrix, and nanoclay; The photoinitiator includes one or more of Irgacure 2959, phenyl-2,4,6-trimethylbenzoyl lithium phosphite, 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; And / or, the cells in the cell suspension include one or more of fibroblasts, vascular endothelial cells, epithelial keratinocytes, nerve cells, muscle cells, chondrocytes, osteoblasts, intestinal epithelial cells, hepatocytes, lung epithelial cells, immune cells, muscle stem cells, embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, epidermal stem cells, adipose stem cells, stem cells and functional cells differentiated from the above cells, organoid suspensions extracted from various normal or cancerous tissues and completed after subculture, and exosome vesicles secreted by cells.

6. The method for preparing a multi-material cell-laden 3D scaffold according to claim 1, characterized in that: In the step S2, the designed model includes multiple structures, and the multiple structures are combined into the model; Each of the structures is printed using a printing ink.

7. The method for preparing a multi-material cell-laden 3D scaffold according to claim 6, characterized in that: The 3D printing device has a material tank, and before the 3D bracket is printed, the printing ink is placed in the material tank; The number of the material trough is one or more; When there is only one material tank, after one structure is printed, the printing device is paused, the printing ink in the material tank is replaced with the printing ink required for another structure, and the residual printing ink in the 3D bracket that has been printed on the printing platform is cleaned and put back to the original position of the device, and then the pause of the printing device is canceled and the structure printing is continued; When there are multiple material tanks, each type of printing ink is placed in a corresponding material tank. During the process of printing a 3D bracket by the 3D printing device, the printing ink in the corresponding material tank is automatically selected or switched to print the 3D bracket. Before switching, negative pressure suction and / or self-rotation drying of the printing platform are automatically used to clean the residual printing ink in the 3D bracket that has been printed on the printing platform.

8. A multi-material cell-laden 3D scaffold, characterized by: It is prepared by the preparation method of the multi-material cell-laden 3D scaffold according to any one of claims 1 to 7, The printing ink used in the inlet and outlet is the same as or different from the printing ink of the main body; The cell-carrying 3D scaffold comprises a main body having cells and microchannels; The main body is provided with an inlet and an outlet, and two ends of the microchannel are respectively connected with the inlet and the outlet; And / or, the main body has at least one liquid storage groove, and both ends of the microchannel are respectively connected to one or more of the liquid storage grooves; And / or, the main body has at least two support rods, and the support rods are connected to the stretching device.

9. An application method of a multi-material cell-laden 3D scaffold, characterized in that: The 3D scaffold is placed in a culture dish, a culture medium is added to the culture dish or the storage tank of the 3D scaffold, and the culture dish is placed in a carbon dioxide incubator to provide a dynamic culture or test environment for the 3D scaffold; The 3D scaffold is prepared by the preparation method of the multi-material cell-laden 3D scaffold as described in any one of claims 1 to 7.

10. The application method of the multi-material cell-laden 3D scaffold according to claim 9, characterized in that: The 3D support has an inlet and an outlet, and the 3D support has a microchannel connected to the inlet and the outlet. The 3D support is placed in a culture dish, and the culture tube is connected to the pre-printed inlet and outlet. The peristaltic pump, the syringe pump or the air pump is connected to the liquid reservoir through the culture tube. After the 3D support is placed in a carbon dioxide incubator, the peristaltic pump, the syringe pump or the air pump is turned on. The peristaltic pump, the syringe pump or the air pump pumps the culture medium in the liquid reservoir into the inlet of the 3D support through the culture tube, and the culture medium flows out from the outlet of the 3D support after passing through the microchannel in the 3D support, thereby realizing the circulation of the culture medium in the formed 3D support and providing a dynamic culture environment for the 3D support. And / or, the outer side of the 3D scaffold has at least one liquid storage tank, and the inside of the 3D scaffold has a microchannel connected to the liquid storage tank. After step S2, the 3D scaffold is placed in a culture dish, the culture medium is poured into the liquid storage tank, the culture dish carrying the 3D scaffold is placed on a shaker, and the shaker is placed in a carbon dioxide incubator. The shaker is turned on and moved, and the culture medium in the liquid storage tank circulates or reciprocates through the microchannel due to the action of gravity or centrifugal force, so that the culture medium circulates in the molded 3D scaffold, providing a dynamic culture environment for the 3D scaffold; And / or, the outer end of the 3D scaffold has at least two support rods, and the support rods are connected to the stretching device. After step S2, the 3D scaffold is placed in a culture dish, and then the culture medium is poured into the culture dish. The culture dish carrying the 3D scaffold and the stretching device connected to the support rods are placed in a carbon dioxide incubator together, and the stretching device is turned on and moved, so that the 3D scaffold reciprocates under the stretching force, providing a mechanical dynamic culture environment for the 3D scaffold.

11. The application method of the multi-material cell-laden 3D scaffold according to claim 10, characterized in that: After the cell culture is completed or the 3D scaffold is directly printed using the cultured cells, the culture medium is replaced with the test object, and the above steps are repeated to provide a dynamic test environment for the 3D scaffold.

Images