CN108274734B - System and method for manufacturing in-vitro 3D tumor microsphere model with controllable core-shell structure - Google Patents
System and method for manufacturing in-vitro 3D tumor microsphere model with controllable core-shell structure Download PDFInfo
- Publication number
- CN108274734B CN108274734B CN201711312531.4A CN201711312531A CN108274734B CN 108274734 B CN108274734 B CN 108274734B CN 201711312531 A CN201711312531 A CN 201711312531A CN 108274734 B CN108274734 B CN 108274734B
- Authority
- CN
- China
- Prior art keywords
- microsphere
- iii
- shell
- material injection
- injection unit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G09—EDUCATION; CRYPTOGRAPHY; DISPLAY; ADVERTISING; SEALS
- G09B—EDUCATIONAL OR DEMONSTRATION APPLIANCES; APPLIANCES FOR TEACHING, OR COMMUNICATING WITH, THE BLIND, DEAF OR MUTE; MODELS; PLANETARIA; GLOBES; MAPS; DIAGRAMS
- G09B23/00—Models for scientific, medical, or mathematical purposes, e.g. full-sized devices for demonstration purposes
- G09B23/28—Models for scientific, medical, or mathematical purposes, e.g. full-sized devices for demonstration purposes for medicine
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y10/00—Processes of additive manufacturing
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y30/00—Apparatus for additive manufacturing; Details thereof or accessories therefor
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
- B29L—INDEXING SCHEME ASSOCIATED WITH SUBCLASS B29C, RELATING TO PARTICULAR ARTICLES
- B29L2031/00—Other particular articles
- B29L2031/40—Test specimens ; Models, e.g. model cars ; Probes
Abstract
The invention discloses a system and a method for manufacturing an in-vitro 3D tumor microsphere model with a controllable core-shell structure. The system comprises: the method comprises the following steps of combining a mold and a biological material pouring platform, wherein the used mold is directly formed by a 3D printer according to an STL file; the biomaterial perfusion platform is combined with a mould combination, and a micro pump is used for realizing the precise perfusion process of the material. The invention is based on a 3D printing mold, and by means of sequential combination of different molds, by injecting biological materials into different mold cavities, the biological materials, cells, medicines or any combination of the above materials injected into the molds are formed into the tumor in vitro 3D microsphere model with controllable and accurate internal structure and morphological characteristics.
Description
Technical Field
The invention relates to a high-throughput and high-repeatability manufacturing system and method of in-vitro 3D tumor microspheres, in particular to a manufacturing system and method of an in-vitro 3D tumor microsphere model with a controllable core-shell structure, which can realize that the microspheres have the controllable core-shell structure, the material components of the core-shell can be biological materials, cells, medicaments or any combination of the components, and the constructed microspheres can be applied to the related pathology and pharmacological analysis of tumors.
Background
Cancer has become the first cause of death in humans in addition to cardiovascular and cerebrovascular diseases. According to data published by international cancer research institutes under the world health organization, the global cancer incidence is increasing at an alarming rate, and in the next two decades, new cases of cancer may reach 2200 million per year and the number of deaths may reach 1300 million.
Tumors grow in vivo in a three-dimensional pattern, with tumor cells not only contacting each other, but also their cells and the extracellular matrix, whose biological behavior is influenced by a number of factors in vivo in common. The functions of cell secretion, adhesion, invasion, metastasis and the like can be obviously influenced by the microenvironment of the tumor cells, the interaction among cells and the interaction between the cells and the matrix, so how to effectively analyze and evaluate the behavior rule of the tumor cells in vivo has important significance for effectively disclosing the pathological process of the tumor. On the other hand, in recent years, many targeted drugs, such as imatinib, trastuzumab, crizotinib, vemurafenib and the like, begin to show application potential in tumor treatment, but because the drugs enter the body or reach tumors, the drugs are often affected by signal exchange inside cancer cells, interaction between the cancer cells and other types of cells, and tumor microenvironment on the tumor cells, the analysis of the action mechanism of the drugs is very difficult, and the research and application of novel tumor treatment drugs are severely limited. Under the background, 3D cell microsphere models which can effectively simulate the tumor cell survival microenvironment and can reflect the interaction between cells and matrix are increasingly focused and valued.
At present, the manufacturing method of the 3D tumor microsphere mainly includes: the hanging drop method, the micro-porous plate method, the micro-carrier method, the bottle rotating method and the micro-fluidic method. Although 3D cell microspheres can be obtained by the methods at present, the precise and controllable distribution of materials in the 3D microspheres in space cannot be effectively realized, and each process has great limitation on the applicable range of the materials. In addition, when the cell microspheres are constructed by a plurality of methods at present, the high repeatability of the manufacturing process cannot be effectively ensured, and the changes of the geometric forms and the sizes of the 3D microsphere individuals can cause the differences of the influence rules of the microenvironment among cells, between cells and matrixes and between the survival microenvironments of the cells, so that the constructed microspheres have the defects in the aspects of detecting the repeatability and the reliability of the structure when being used as pathological or pharmacological analysis samples, and the application and the popularization of the microspheres are directly limited.
The invention provides a mold-based microsphere manufacturing method aiming at the current research situation and the existing problems, which ensures that the constructed 3D tumor microsphere model can have a controllable core-shell layer structure, and the material components of the core-shell layer can be biological materials, cells, medicines or any combination of the components. Because the layer thickness of the cell microsphere core shell layer constructed by the method is controllable, and the distribution of the material components is controllable, the constructed microsphere can effectively simulate the behavior rule of the tumor cells under the environments with different mechanical strengths and different biochemical component gradient changes, and simultaneously has the condition of analyzing the interaction between multiple cells and the interaction between the cells and the extracellular matrix, thereby being a novel method with great potential.
Disclosure of Invention
The invention aims to provide a system and a method for manufacturing an in-vitro 3D tumor microsphere model with a controllable core-shell structure aiming at the defects of the prior art, which mainly comprise a multi-layer microsphere manufacturing mould with a new shell structure manufactured by a 3D printing technology; the thickness and shape of the microsphere core-shell layer can be accurately controlled by pouring the required biological material, cells, medicaments or any combination of the above materials into the die cavity in the die and depending on the shape and structure of the die cavity. The method has the characteristic of obvious repeatability in manufacturing, and lays a solid foundation for constructing a large number of 3D tumor microsphere models with uniform quality.
In order to achieve the purpose, the invention adopts the following technical scheme:
the utility model provides an external 3D microballon model manufacturing system of tumour with controllable core-shell structure, includes microballon mould combination, material perfusion and shaping platform, its characterized in that: the material filling and forming platform consists of a micro pump executing mechanism, a sterilizing injector, a connecting pipe and a micro pump controller; the sterilizing injector is fixedly arranged on the micro pump executing mechanism, the micro pump executing mechanism pushes the sterilizing injector to accurately supply materials under the control of the micro pump controller, the outlet of the sterilizing injector is connected with a connecting pipe, the connecting pipe is connected with the microsphere mold combination, and an ultraviolet lamp is arranged above the microsphere mold combination.
The microsphere mold combination is a single-flux microsphere mold combination and comprises a microsphere forming left half mold, a microsphere forming right half mold, a microsphere core mold, an inlet pipe and an outlet pipe, wherein an injection opening in the microsphere forming left half mold is connected with the inlet pipe, and an exhaust opening in the microsphere forming right half mold is connected with the outlet pipe. The microsphere forming left half die, the microsphere forming right half die and the microsphere core die are matched, and the microsphere shell layer with the cylindrical channel of the 3D microsphere can be formed. After the shell layer is solidified and formed, the shell layer solidified after the microsphere core mold is taken down and used as a cavity for forming the core layer of the microsphere and the residual shell layer to be sealed.
The microsphere mold combination is a high-throughput microsphere mold combination, so that the manufacturing system is expanded into a high-throughput tumor in-vitro 3D microsphere model manufacturing system, and the high-throughput manufacturing of the tumor in-vitro 3D microsphere model is realized; the high-throughput microsphere mold combination comprises a microsphere forming unit left half mold, a microsphere forming unit right half mold, a shell material injection unit, an exhaust unit, a shell forming unit, a core layer material injection unit, a shell sealing material injection unit and a splitter, wherein the microsphere forming unit is formed by splicing the microsphere forming unit left half mold and the microsphere forming unit right half mold, the microsphere forming units can be spliced with each other, and the number of microspheres obtained by one-time injection is controlled; the shell material injection unit is provided with a positioning pin which is matched with the positioning holes on the microsphere forming unit and the exhaust unit so as to ensure the assembly precision; the bottom surface of the shell layer forming unit is provided with a cylindrical array, the bottom surface of the cylinder is hemispherical, and the cylindrical array is matched with the spherical cavity of the microsphere forming unit to form a shell layer die; runners are arranged in the core layer material injection unit and the shell layer sealing material injection unit, the bottoms of the core layer material injection unit and the shell layer sealing material injection unit are cylindrical arrays, the core layer material injection unit and the shell layer sealing material injection unit are aligned to a spherical cavity of the microsphere forming unit after die assembly, a pore passage is reserved in the cylinder and connected with the upper runner, and a plurality of material injection ports are reserved at the top of the core layer material injection unit and the shell layer sealing material injection; a flow channel is arranged in the flow divider and divides one path of input material into multiple paths of output; the material is poured and is formed the platform for high flux material is poured and is formed the platform, and this high flux material is poured and is formed the platform and include: the device comprises a micro pump actuating mechanism, a sterilization injector, a connecting pipe and a micro pump controller; the sterilizing injector is fixedly arranged on the micro pump actuating mechanism, and the micro pump actuating mechanism pushes the sterilizing injector to accurately supply materials under the control of the micro pump controller. The outlet of the sterilizing injector is connected with the connecting pipe. When the shell layer of the microsphere is formed, the connecting pipe is connected with the shell layer material injection unit, the material in the sterilizing injector is extruded under the pushing of the micro pump actuating mechanism and is injected from the inlet of the shell layer material injection unit through the connecting pipe; when the microsphere core layer is formed, the connecting pipe is connected with the inlet of the flow divider, the outlet of the flow divider is connected with the core layer material injection unit, materials in the sterilization injector are extruded under the pushing of the micro pump actuating mechanism, enter the flow divider for flow division through the connecting pipe, then enter the core layer material injection unit through a multi-channel pipeline, and finally align the microsphere forming unit for material injection; in the closed forming of the microsphere shell, the connecting pipe is connected with the inlet of the flow divider, the outlet of the flow divider is connected with the shell sealing material injection unit, materials in the sterilization injector are extruded under the pushing of the micro pump actuating mechanism, enter the flow divider for flow dividing through the connecting pipe, then enter the shell sealing material injection unit through a multi-way pipeline, and finally align the microsphere forming unit for material injection.
1) Manufacturing molds for constructing the core-shell structured microspheres are manufactured by 3D printing technology, the molds described herein are directly manufactured by a commercial 3D printer, and the design of the molds specifically comprises 3 submolds, as shown in fig. 2. After the microsphere forming left half die (I1) and the microsphere forming right half die (I2) are closed, a cavity with a round hole at the top is formed, an injection port (provided with an inlet pipe (I4)) and an exhaust port (provided with an outlet pipe (I5)) are respectively arranged at two sides, and a cylindrical core is fixed at the bottom of the microsphere core die (I3) and used for forming a cylindrical channel in the middle of a microsphere so as to allow the injection of a core layer material in a subsequent process.
2) Forming a shell layer of the 3D microsphere based on the mold: the shaping here is based on the use of a material infusion system consisting of a micropump, a disposable syringe, a connecting tube and the above-mentioned mould, as shown in fig. 1. After a microsphere forming left half die (I1), a microsphere forming right half die (I2) and a microsphere core die (I3) are closed, a syringe piston is pushed through a micro pump, required biological materials, cells, medicines or any combination of the materials are injected from an inlet pipe (I4) of an injection port of the microsphere forming left half die (I1) through an inlet connecting pipe until the materials flow out from an outlet pipe (I5) of an exhaust port of the microsphere forming right half die (I2), after the materials are filled in a shell cavity in the die, and then the cross-linking and solidification of the materials are realized by adjusting the temperature of the die or loading illumination (ultraviolet, white light and blue light) according to the solidification characteristics (photosensitive or temperature sensitive) of the selected materials; and after the materials are solidified, taking down the microsphere core mold (I3) to obtain the shell of the 3D microsphere with the cylindrical channel.
3) Forming a core layer of the 3D microsphere based on the mold: and aligning and connecting the connecting pipe with the obtained cylindrical channel on the 3D microsphere shell, then pushing the piston of the injector by using a micro pump, and injecting a material into the microsphere core layer to form the microsphere core layer.
4) Closing cylindrical channels at the tops of the microspheres: after the microsphere core layer is formed, the connecting pipe is connected with the cylindrical channel in an aligning mode, a micro pump in the filling system pushes an injector piston to inject materials which are the same as the shell layer into the cylindrical channel through the connecting pipe according to set parameters of a controller, and then cross-linking curing of the materials is achieved by adjusting the temperature of a mould or loading illumination (ultraviolet, white light and blue light) according to curing characteristics (photosensitive or temperature sensitive) of the selected materials, and finally the complete microsphere with the core-shell layer structure is formed.
The invention can be expanded from a single mold cavity to a multi-mold cavity as required, as shown in fig. 4, thereby realizing high-throughput preparation of microspheres. The preparation of the high-flux microspheres adopts the following technical scheme:
1) the manufacturing mold for constructing the core-shell structured microspheres was manufactured by 3D printing technology, and the mold described herein was directly manufactured by a commercial 3D printer, as shown in fig. 4. After the left half die (III 1) and the right half die (III 2) of the microsphere forming unit are assembled, a single row of microsphere forming unit (III 3) with a spherical cavity is formed, a plurality of microsphere forming units are arranged in series, positioning holes in the forming units are kept aligned, a shell material injection unit (III 4) and an exhaust unit (III 5) are installed to complete the assembly of the die, and the assembly accuracy of the shell material injection unit (III 4) and the shell material injection unit (III 3) is guaranteed by positioning pins in the shell material injection unit (III 4) and positioning holes in the microsphere forming unit (III 3) and the exhaust unit (III 5).
2) Forming a shell layer of the 3D microsphere based on the mold: the shaping here is based on the use of a material infusion system consisting of a micro pump, a disposable syringe, a connecting tube and the above mentioned mould, as shown in fig. 3 a. After a microsphere forming unit (III 3), a shell material injection unit (III 4), an exhaust unit (III 5) and a shell forming unit (III 6) are assembled, a material inlet of the shell material injection unit (III 4) is connected with a sterilization injector (II 2) through a connecting pipe (II 3), an injector piston is pushed through a micro pump, a required biological material, cells, a medicine or any combination of the above materials are injected through the shell material injection unit (III 4) until the materials flow out from the exhaust unit (III 5), and after the materials are filled in a shell cavity in a mold, the crosslinking and curing of the materials are realized by adjusting the temperature of the mold or loading illumination (ultraviolet, white light and blue light) according to the curing characteristics (photosensitive or temperature sensitive) of the selected materials; and (5) after the materials are solidified, taking down the shell layer forming unit (III 6) to obtain the shell of the 3D microsphere with the cylindrical channel.
3) Based on the above mold, the core layer of the 3D microspheres is formed, as shown in fig. 3 b: the method comprises the steps of combining a microsphere forming unit (III 3), a shell material injection unit (III 4), an exhaust unit (III 5) and a core layer material injection unit (III 7), connecting a flow divider (III 9) inlet and a sterilization injector (II 2) through a connecting pipe (II 3), connecting a flow divider (III 9) multi-path outlet and a material injection port of the core layer material injection unit (III 7), then pushing an injector piston through a micro pump, injecting a material to form a core layer of microspheres, and taking down the core layer material injection unit (III 7) to obtain a microsphere core layer.
4) Closing cylindrical channels at the tops of the microspheres: after the microsphere core layer is formed, a microsphere forming unit (III 3), a shell material injection unit (III 4), an exhaust unit (III 5) and a shell sealing material injection unit (III 8) are assembled, an inlet of a flow divider (III 9) is connected with a sterilization injector (II 2) through a connecting pipe (II 3), a multi-way outlet of the flow divider (III 9) is connected with a material injection port of the shell sealing material injection unit (III 8), materials are injected, then the curing characteristic (photosensitive or temperature sensitive) of the selected materials is aimed at, and the crosslinking and curing of the materials are realized by adjusting the temperature of a mold or loading the mode of illumination (ultraviolet, white light and blue light), and finally the complete microsphere with the core-shell layer structure is formed.
Compared with the prior art, the invention has the following obvious and prominent substantive characteristics and remarkable advantages:
1) the 3D printing technology is utilized to manufacture the mold for forming the 3D microspheres, so that the manufacturing period is short;
2) the design of the mould can be adjusted according to the actual structure and cell distribution condition of different tumor tissues, so that the 3D microspheres with different core-shell structures (the thickness of the core-shell layer is adjustable, and the distribution of material components is adjustable) are manufactured.
3) High reproducibility of the manufacture of microspheres can be achieved.
4) High throughput manufacturing of microspheres can be achieved by expanding the mold.
Drawings
FIG. 1 is a schematic diagram of a single-throughput microsphere-forming material infusion and forming system.
FIG. 2 is a schematic view of a 3D microsphere mold assembly.
FIG. 3 is a schematic diagram of the platform composition for perfusion and formation of a high throughput microsphere-forming material.
Fig. 4 is a schematic diagram of the assembly and disassembly of high throughput 3D microsphere molds.
Detailed Description
The structure of the preferred embodiment of the invention is detailed as follows:
the first embodiment is as follows:
referring to fig. 1-4, the system for manufacturing the tumor in vitro 3D microsphere model with the controllable core-shell structure comprises a microsphere mold assembly, a material perfusion and forming platform, and is characterized in that: the material filling and forming platform consists of a micro pump actuating mechanism (II 1), a sterilizing injector (II 2), a connecting pipe (II 3) and a micro pump controller (II 4); the sterilizing injector (II 2) is installed and fixed on the micro pump executing mechanism (II 1), the micro pump executing mechanism (II 1) pushes the sterilizing injector (II 2) to accurately supply materials under the control of a micro pump controller (II 4), the outlet of the sterilizing injector (II 2) is connected with a connecting pipe (II 3), the connecting pipe (II 3) is connected with a microsphere mold combination (I), and an ultraviolet lamp (II 5) is installed above the microsphere mold combination (I).
Example two:
referring to fig. 1 and fig. 2, the present embodiment is substantially the same as the first embodiment, and the features are as follows: the microsphere mold combination is a single-flux microsphere mold combination (I) and comprises a microsphere forming left half mold (I1), a microsphere forming right half mold (I2), a microsphere core mold (I3), an inlet pipe (I4) and an outlet pipe (I5), wherein an injection opening in the microsphere forming left half mold (I1) is connected with the inlet pipe (I4), and an exhaust opening in the microsphere forming right half mold (I2) is connected with the outlet pipe (I5). And the microsphere forming left half die (I1), the microsphere forming right half die (I2) and the microsphere core die (I3) are combined, so that a microsphere shell layer with a cylindrical channel of the 3D microsphere can be formed. After the shell is solidified and formed, the solidified shell after the mould 3 is taken down is taken as a cavity to form a core layer of the microsphere and the shell to be sealed.
Example three:
referring to fig. 3 and fig. 4, this embodiment is substantially the same as the first embodiment, and is characterized in that the microsphere mold assembly is a high-throughput microsphere mold assembly (iii), so that the manufacturing system is expanded to a high-throughput tumor in vitro 3D microsphere model manufacturing system, thereby realizing high-throughput manufacturing of the tumor in vitro 3D microsphere model; the high-throughput microsphere mold combination (III) comprises a microsphere forming unit left half mold (III 1), a microsphere forming unit right half mold (III 2), a shell material injection unit (III 4), an exhaust unit (III 5), a shell forming unit (III 6), a core layer material injection unit (III 7), a shell sealing material injection unit (III 8) and a flow divider (III 9), wherein the microsphere forming unit left half mold (III 1) and the microsphere forming unit right half mold (III 2) are spliced to form a microsphere forming unit (III 3), the microsphere forming units (III 3) can be spliced with each other, and the number of microspheres obtained by one-time perfusion is controlled; runners are arranged in the shell material injection unit (III 4) and the exhaust unit (III 5) to facilitate material injection and removal, and the shell material injection unit (III 4) is provided with a positioning pin which is matched with positioning holes in the microsphere forming unit (III 3) and the exhaust unit (III 5) to ensure assembly accuracy; the bottom surface of the shell layer forming unit (III 6) is provided with a cylindrical array, the bottom surface of the cylinder is hemispherical, and the cylindrical array is matched with the spherical cavity of the microsphere forming unit (III 3) to form a shell layer die; runners are arranged in the core layer material injection unit (III 7) and the shell layer sealing material injection unit (III 8), the bottoms of the core layer material injection unit and the shell layer sealing material injection unit are cylindrical arrays, the core layer material injection unit and the shell layer sealing material injection unit are aligned to a spherical cavity of the microsphere forming unit (III 3) after die assembly, a pore channel is reserved in the cylinder and connected with the upper runner, and a plurality of material injection ports are reserved at the top of the core layer material injection unit and the shell layer sealing material injection unit; a flow channel is arranged in the flow divider (III 9) and divides one path of input material into multiple paths of output; the material pouring and forming platform is a high-flux material pouring and forming platform (IV) which comprises: a micro pump actuating mechanism (II 1), a sterilizing injector (II 2), a connecting pipe (II 3) and a micro pump controller (II 4); the sterilizing injector (II 2) is fixedly arranged on the micro pump actuating mechanism (II 1), and the micro pump actuating mechanism (II 1) pushes the sterilizing injector (II 2) to accurately supply materials under the control of the micro pump controller (II 4). The outlet of the sterilizing injector (II 2) is connected with a connecting pipe (II 3). When the shell layer of the microsphere is formed, the connecting pipe (II 3) is connected with the shell layer material injection unit (III 4), the material in the sterilizing injector (II 2) is extruded under the pushing of the micro pump actuating mechanism (II 1), and is injected from the inlet of the shell layer material injection unit (III 4) through the connecting pipe (II 3); when the microsphere core layer is formed, the connecting pipe (II 3) is connected with an inlet of the flow divider (III 9), an outlet of the flow divider (III 9) is connected with the core layer material injection unit (III 7), materials in the sterilizing injector (II 2) are extruded under the pushing of the micro pump actuating mechanism (II 1), enter the flow divider (III 9) through the connecting pipe (II 3) for flow division, then enter the core layer material injection unit (III 7) through a multi-channel pipeline, and finally align the microsphere forming unit (III 3) for material injection; in the closed forming of a microsphere shell layer, a connecting pipe (II 3) is connected with an inlet of a flow divider (III 9), an outlet of the flow divider (III 9) is connected with a shell layer closed material injection unit (III 8), materials in a sterilization injector (II 2) are extruded under the pushing of a micro pump actuating mechanism (II 1), enter the flow divider (III 9) for flow division through the connecting pipe (II 3), then enter the shell layer closed material injection unit (III 8) through a multi-way pipeline, and finally align the microsphere forming unit (III 3) for material injection.
Example four:
the method for preparing the 3D microsphere model constructed by the biological material comprises the following operation steps:
1) preparation of test materials: the biomaterial for filling the 3D microsphere core layer is gelatin (chemical purity CP, national medicine): dissolving gelatin in water to obtain 20 wt% solution, sterilizing, and mixing with gelatin solution containing 2 × 106mL-1RT4 cell (shanghai cell bank of chinese academy of sciences) suspension according to 1: 1 proportion to obtain 10 wt% of RT4 with the cell density of 1 multiplied by 106mL-1The cell gelatin blend solution of (a); the biological material used for filling the 3D microsphere shell is gelatin (chemical purity CP, Chinese medicine): dissolving gelatin in deionized water to prepare a solution with the mass fraction of 14%.
2) Manufacturing a mould: designing models of 3 moulds through three-dimensional modeling software, converting the models into STL format files, inputting the STL format files into a photocuring 3D printer, printing the moulds, and performing sterilization treatment after printing. The mold material used was a photosensitive resin, and the resulting mold was as shown in FIG. 2.
3) Forming a shell layer of the 3D microsphere with the cylindrical channel: preheating a prepared gelatin (14%) solution, preserving heat, filling the solution into a sterilization injector (II 2), fixing the sterilization injector (II 2) on a micro pump actuating mechanism (II 1), connecting an outlet of the sterilization injector (II 2) with a connecting pipe (II 3), combining a microsphere forming left half die (I1), a microsphere forming right half die (I2) and a microsphere core die (I3), installing an inlet pipe (I4) and an outlet pipe (I5) after die assembly, and connecting the connecting pipe (II 3) with the inlet pipe (I4). Setting the feeding flow of a micro pump controller (II 4) to be 30 mu L/min, driving a micro pump actuating mechanism (II 1) by the micro pump controller (II 4) to inject gelatin solution into a mold from an inlet pipe (I4) arranged on a microsphere forming left half mold (I1) until the solution flows out from an outlet pipe (I5) of a microsphere forming right half mold (I2), then curing at low temperature, and taking down a microsphere core mold (I3) after the curing is finished, so that a shell layer of the 3D microsphere with a cylindrical channel can be obtained.
4) Core layer of formed 3D microspheres: preheating the prepared gelatin RT4 cell blending solution, preserving heat, loading into a sterilization injector (II 2), fixing on a micro pump actuating mechanism (II 1), connecting an outlet of the sterilization injector (II 2) with a connecting pipe (II 3), aligning and connecting the connecting pipe (II 3) with a cylindrical channel at the upper end of a formed microsphere, setting the feeding flow of a micro pump controller (II 4) to be 1 muL/min, driving the micro pump actuating mechanism (II 1) by the micro pump controller (II 4) to inject the gelatin solution into the cylindrical channel from the connecting pipe (II 3), and controlling the feeding duration to be 1 s. And (4) taking down the connecting pipe (II 3), and curing at low temperature to obtain the microsphere core layer.
5) Closing cylindrical channels at the tops of the microspheres: preheating and insulating the prepared gelatin solution, filling the gelatin solution into a sterilization injector (II 2), fixing the sterilization injector (II 2) on a micro pump actuating mechanism (II 1), connecting an outlet of the sterilization injector (II 2) with a connecting pipe (II 3), aligning and connecting the connecting pipe (II 3) with a cylindrical channel at the upper end of the formed microsphere, setting the feed flow of a micro pump controller (II 4) to be 1 muL/min, driving the micro pump actuating mechanism (II 1) by the micro pump controller (II 4) to inject the gelatin solution into the cylindrical channel from the connecting pipe (II 3), and sealing the channel. And (4) taking down the connecting pipe (II 3), curing at low temperature, and obtaining a complete microsphere shell after the material is cured.
Example five:
the method for preparing the 3D microsphere model constructed by the biomaterial by using the high-throughput mould comprises the following operation steps:
1) preparation of test materials: the biomaterial for filling the 3D microsphere core layer is gelatin (chemical purity CP, national medicine): dissolving gelatin in water to obtain 20 wt% solution, sterilizing, and mixing with gelatin solution containing 2 × 106mL-1RT4 cell (shanghai cell bank of chinese academy of sciences) suspension according to 1: 1 proportion to obtain 10 wt% of RT4 cellsDensity 1X 106mL-1The cell gelatin blend solution of (a); the biological material used for filling the 3D microsphere shell is gelatin (chemical purity CP, Chinese medicine): dissolving gelatin in deionized water to prepare a solution with the mass fraction of 14%.
2) Manufacturing a mould: designing a mold model through three-dimensional modeling software, converting the model into a file in an STL format, inputting the file into a photocuring 3D printer, printing the mold, and performing sterilization treatment after printing. The mold material used was a photosensitive resin, and the resulting mold was as shown in fig. 4.
3) Forming a shell layer of the 3D microsphere with the cylindrical channel: preheating a prepared gelatin (14%) solution, preserving heat, loading into a sterilization injector (II 2), fixing on a micro pump actuating mechanism (II 1), connecting an outlet of the sterilization injector (II 2) with a connecting pipe II 3, combining a plurality of microsphere forming units (III 3) and a shell material injection unit (III 4) which are arranged in series, assembling an exhaust unit (III 5) as a female die and then with a shell forming unit (III 6), clamping by using a clamp, and connecting the connecting pipe (II 3) with an inlet of the shell material injection unit (III 4). Setting the feeding flow rate of the micro-pump controller (II 4) to be 0.3mL/min, driving the micro-pump actuator (II 4) by the micro-pump controller (II 4) to inject the gelatin solution into the mould from the inlet of the shell material injection unit (III 4) until the solution flows out of the exhaust hole of the exhaust unit (III 5), then solidifying at low temperature, and taking down the shell forming unit (III 6) after completion, thus obtaining the shell of the 3D microsphere with the cylindrical channel.
4) Core layer of formed 3D microspheres: preheating a prepared gelatin RT4 cell blending solution, preserving heat, filling into a sterilization injector (II 2), fixing on a micro pump actuating mechanism (II 1), connecting an outlet of the sterilization injector (II 2) with a connecting pipe (II 3), connecting the connecting pipe (II 3) with an inlet of a shunt (III 9), connecting a multi-path outlet of the shunt (III 9) with an injection opening at the top of a core layer material injection unit (III 7), setting the feeding flow of a micro pump controller (II 4) to be 0.1mL/min, and driving the micro pump actuating mechanism (II 1) by the micro pump controller (II 4) to inject the gelatin solution into a spherical chamber of a microsphere forming unit (III 3) from a cylindrical array pore passage at the bottom of the core layer material injection unit (III 7). And taking down the core layer material injection unit (III 7), and curing at low temperature to obtain the microsphere core layer.
5) Closing cylindrical channels at the tops of the microspheres: preheating and insulating the prepared gelatin solution, filling the gelatin solution into a sterilization injector (II 2), fixing the sterilization injector (II 2) on a micro pump actuating mechanism (II 1), connecting an outlet of the sterilization injector (II 2) with a connecting pipe (II 3), connecting the connecting pipe (II 3) with an inlet of a shunt (III 9), connecting a multi-path outlet of the shunt (III 9) with an injection port at the top of a shell sealing material injection unit (III 8), setting the feeding flow of a micro pump controller (II 4) to be 0.1mL/min, driving the micro pump actuating mechanism (II 1) by the micro pump controller (II 4) to inject the gelatin solution into a spherical chamber of the microsphere forming unit (III 3) from a cylindrical array pore channel at the bottom of the shell sealing material injection unit (III 8), and sealing a microsphere shell. And taking down the shell layer sealing material injection unit (III 8), solidifying the gelatin at low temperature, and obtaining the complete microsphere shell after solidification.
Claims (5)
1. The utility model provides an external 3D tumour microballon model manufacturing system with controllable core shell structure, includes microballon mould combination, material perfusion and shaping platform, its characterized in that: the material filling and forming platform consists of a micro pump actuating mechanism (II 1), a sterilizing injector (II 2), a connecting pipe (II 3) and a micro pump controller (II 4); the sterilizing injector (II 2) is installed and fixed on the micro pump executing mechanism (II 1), the micro pump executing mechanism (II 1) pushes the sterilizing injector (II 2) to accurately supply materials under the control of a micro pump controller (II 4), the outlet of the sterilizing injector (II 2) is connected with a connecting pipe (II 3), the connecting pipe (II 3) is connected with a microsphere mold combination (I), and an ultraviolet lamp (II 5) is installed above the microsphere mold combination (I).
2. The in vitro 3D tumor microsphere model manufacturing system with controllable core-shell structure according to claim 1, characterized in that: the microsphere mold combination is a single-flux microsphere mold combination (I) and comprises a microsphere forming left half mold (I1), a microsphere forming right half mold (I2), a microsphere core mold (I3), an inlet pipe (I4) and an outlet pipe (I5), wherein an injection port on the microsphere forming left half mold (I1) is connected with the inlet pipe (I4), and an exhaust port on the microsphere forming right half mold (I2) is connected with the outlet pipe (I5); the microsphere forming left half die (I1), the microsphere forming right half die (I2) and the microsphere core die (I3) are combined, and a microsphere shell layer with a cylindrical channel of a 3D microsphere can be formed; after the shell layer is solidified and formed, the shell layer solidified after the microsphere core mould (I3) is taken down is used as a core layer and a residual shell layer to be sealed of a cavity which can be used for forming the microsphere.
3. The in vitro 3D tumor microsphere model manufacturing system with controllable core-shell structure according to claim 1, characterized in that: the microsphere mold combination is a high-throughput microsphere mold combination (III), so that the manufacturing system is expanded into a high-throughput tumor in-vitro 3D microsphere model manufacturing system, and the high-throughput manufacturing of the tumor in-vitro 3D microsphere model is realized; the high-throughput microsphere mold combination (III) comprises a microsphere forming unit left half mold (III 1), a microsphere forming unit right half mold (III 2), a shell material injection unit (III 4), an exhaust unit (III 5), a shell forming unit (III 6), a core layer material injection unit (III 7), a shell sealing material injection unit (III 8) and a flow divider (III 9), wherein the microsphere forming unit left half mold (III 1) and the microsphere forming unit right half mold (III 2) are spliced to form a microsphere forming unit (III 3), the microsphere forming units (III 3) can be spliced with each other, and the number of microspheres obtained by one-time perfusion is controlled; runners are arranged in the shell material injection unit (III 4) and the exhaust unit (III 5) to facilitate material injection and removal, and the shell material injection unit (III 4) is provided with a positioning pin which is matched with positioning holes in the microsphere forming unit (III 3) and the exhaust unit (III 5) to ensure assembly accuracy; the bottom surface of the shell layer forming unit (III 6) is provided with a cylindrical array, the bottom surface of the cylinder is hemispherical, and the cylindrical array is matched with the spherical cavity of the microsphere forming unit (III 3) to form a shell layer die; runners are arranged in the core layer material injection unit (III 7) and the shell layer sealing material injection unit (III 8), the bottoms of the core layer material injection unit and the shell layer sealing material injection unit are cylindrical arrays, the core layer material injection unit and the shell layer sealing material injection unit are aligned to a spherical cavity of the microsphere forming unit (III 3) after die assembly, a pore channel is reserved in the cylinder and connected with the upper runner, and a plurality of material injection ports are reserved at the top of the core layer material injection unit and the shell layer sealing material injection unit; a flow channel is arranged in the flow divider (III 9) and divides one path of input material into multiple paths of output; the material pouring and forming platform is a high-flux material pouring and forming platform (IV) which comprises: a micro pump actuating mechanism (II 1), a sterilizing injector (II 2), a connecting pipe (II 3) and a micro pump controller (II 4); the sterilizing injector (II 2) is fixedly arranged on the micro pump actuating mechanism (II 1), and the micro pump actuating mechanism (II 1) pushes the sterilizing injector (II 2) to accurately supply materials under the control of the micro pump controller (II 4); the outlet of the sterilizing injector (II 2) is connected with a connecting pipe (II 3); when the shell layer of the microsphere is formed, the connecting pipe (II 3) is connected with the shell layer material injection unit (III 4), the material in the sterilizing injector (II 2) is extruded under the pushing of the micro pump actuating mechanism (II 1), and is injected from the inlet of the shell layer material injection unit (III 4) through the connecting pipe (II 3); when the microsphere core layer is formed, the connecting pipe (II 3) is connected with an inlet of the flow divider (III 9), an outlet of the flow divider (III 9) is connected with the core layer material injection unit (III 7), materials in the sterilizing injector (II 2) are extruded under the pushing of the micro pump actuating mechanism (II 1), enter the flow divider (III 9) through the connecting pipe (II 3) for flow division, then enter the core layer material injection unit (III 7) through a multi-channel pipeline, and finally align the microsphere forming unit (III 3) for material injection; in the closed forming of a microsphere shell layer, a connecting pipe (II 3) is connected with an inlet of a flow divider (III 9), an outlet of the flow divider (III 9) is connected with a shell layer closed material injection unit (III 8), materials in a sterilization injector (II 2) are extruded under the pushing of a micro pump actuating mechanism (II 1), enter the flow divider (III 9) for flow division through the connecting pipe (II 3), then enter the shell layer closed material injection unit (III 8) through a multi-way pipeline, and finally align the microsphere forming unit (III 3) for material injection.
4. A method for manufacturing an in-vitro 3D tumor microsphere model with a controllable core-shell structure, which is operated by the in-vitro 3D tumor microsphere model manufacturing system with the controllable core-shell structure according to claim 2, and is characterized by comprising the following specific operation steps:
1) forming a shell layer of the 3D microsphere with the cylindrical channel: combining a microsphere forming left half die (I1), a microsphere forming right half die (I2) and a microsphere core die (I3), after die assembly, pushing a syringe piston by a micropump in a filling system according to set parameters of a controller, adopting gelatin solution as a biological material, injecting the biological material from an inlet pipe (I4) of an injection port of the microsphere core die (I3) through an inlet connecting pipe until the material flows out from an outlet pipe (I5) of an exhaust port of the microsphere core die (I3), curing the material after the material is filled in a die cavity, and removing the microsphere core die (I3) after the material is cured, so that a shell of a 3D microsphere with a cylindrical channel can be obtained;
2) core layer of formed 3D microspheres: after the shell of the 3D microsphere with the cylindrical channel is formed, the connecting pipe is connected with the cylindrical channel in an aligning way, a micro pump in a perfusion system pushes a syringe piston according to the set parameters of a controller, the mixture of gelatin and RT4 cells is used as a core layer biological raw material for preparing a core layer material, and the core layer biological raw material is quantitatively injected into the center of the microsphere from the cylindrical channel through an inlet connecting pipe to form a core layer of the microsphere;
3) closing cylindrical channels at the tops of the microspheres: after the microsphere core layer is formed, the connecting pipe is aligned and connected with the cylindrical channel, and a micro pump in the perfusion system pushes a syringe piston to inject the same material as the shell into the cylindrical channel through the inlet connecting pipe according to the set parameters of the controller to seal the cylindrical channel; the complete microsphere shell is formed after the material is cured.
5. A method for manufacturing an in-vitro 3D tumor microsphere model with a controllable core-shell structure, which is operated by the in-vitro 3D tumor microsphere model manufacturing system with the controllable core-shell structure according to claim 3, and is characterized by comprising the following specific operation steps:
1) forming a shell layer of the 3D microsphere: the forming is based on the material filling system composed of a micro pump, a disposable injector, a connecting pipe and the mould; after the microsphere forming unit (III 3), the shell material injection unit (III 4), the exhaust unit (III 5) and the shell forming unit (III 6) are closed, a material inlet of the shell material injection unit (III 4) is connected with the sterilization injector (II 2) through the connecting pipe (II 3), the injector piston is pushed through a micro pump, a gelatin solution is used as a biological material, the biological material is injected through the shell material injection unit (III 4) until the material flows out of the exhaust unit (III 5), after the material is filled in a shell cavity in a mold, and then the crosslinking and curing of the material are realized by adjusting the temperature of the mold or loading illumination according to the curing characteristics of the selected material; after the materials are solidified, taking down the shell layer forming unit (III 6) to obtain a shell of the 3D microsphere with the cylindrical channel;
2) core layer of formed 3D microspheres: closing a microsphere forming unit (III 3), a shell material injection unit (III 4), an exhaust unit (III 5) and a core layer material injection unit (III 7), connecting a flow divider (III 9) inlet and a sterilization injector (II 2) by using a connecting pipe (II 3), connecting a flow divider (III 9) multi-path outlet and a material injection port at the top of the core layer material injection unit (III 7), then pushing an injector piston by using a micro pump, injecting a core layer biological raw material into a core layer forming microsphere by using a mixture of gelatin and RT4 cells as a core layer biological raw material for preparing the core layer material, and taking down the core layer material injection unit (III 7) to obtain a microsphere core layer;
3) closing cylindrical channels at the tops of the microspheres: after the microsphere core layer is formed, the microsphere forming unit (III 3), the shell material injection unit (III 4), the exhaust unit (III 5) and the shell sealing material injection unit (III 8) are assembled, an inlet of the flow divider (III 9) is connected with the sterilization injector (II 2) through the connecting pipe (II 3), a multi-way outlet of the flow divider (III 9) is connected with a material injection port of the shell sealing material injection unit (III 8), materials are injected, then, aiming at the curing characteristics of the selected materials, the cross-linking and curing of the materials are realized by adjusting the temperature of a mould or loading the illumination mode, and finally, the complete microsphere with the core-shell structure is formed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711312531.4A CN108274734B (en) | 2017-12-12 | 2017-12-12 | System and method for manufacturing in-vitro 3D tumor microsphere model with controllable core-shell structure |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711312531.4A CN108274734B (en) | 2017-12-12 | 2017-12-12 | System and method for manufacturing in-vitro 3D tumor microsphere model with controllable core-shell structure |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108274734A CN108274734A (en) | 2018-07-13 |
| CN108274734B true CN108274734B (en) | 2020-04-03 |
Family
ID=62801482
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711312531.4A Active CN108274734B (en) | 2017-12-12 | 2017-12-12 | System and method for manufacturing in-vitro 3D tumor microsphere model with controllable core-shell structure |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108274734B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110042077B (en) * | 2019-04-22 | 2021-10-19 | 清华-伯克利深圳学院筹备办公室 | High-flux culture method of organoid spheres |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101448527A (en) * | 2006-05-19 | 2009-06-03 | 香港大学 | Cell-matrix microspheres, methods for preparation and applications |
| CN106128257A (en) * | 2016-08-16 | 2016-11-16 | 杭州科霖医疗科技有限公司 | A kind of method utilizing rapid prototyping technology to make personalization emulation anatomical model |
| CN106178130A (en) * | 2016-07-10 | 2016-12-07 | 上海大学 | The formation system of bifurcation structure three-dimensional layering intravascular stent and method |
| WO2017093567A1 (en) * | 2015-12-04 | 2017-06-08 | Centre Léon-Bérard | Animal tumour model and production method thereof |
| CN107307905A (en) * | 2016-04-26 | 2017-11-03 | 上海拓飞电气有限公司 | A kind of 3D printing, the method that mould embedding obtains human body brainpan model |
-
2017
- 2017-12-12 CN CN201711312531.4A patent/CN108274734B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101448527A (en) * | 2006-05-19 | 2009-06-03 | 香港大学 | Cell-matrix microspheres, methods for preparation and applications |
| WO2017093567A1 (en) * | 2015-12-04 | 2017-06-08 | Centre Léon-Bérard | Animal tumour model and production method thereof |
| CN107307905A (en) * | 2016-04-26 | 2017-11-03 | 上海拓飞电气有限公司 | A kind of 3D printing, the method that mould embedding obtains human body brainpan model |
| CN106178130A (en) * | 2016-07-10 | 2016-12-07 | 上海大学 | The formation system of bifurcation structure three-dimensional layering intravascular stent and method |
| CN106128257A (en) * | 2016-08-16 | 2016-11-16 | 杭州科霖医疗科技有限公司 | A kind of method utilizing rapid prototyping technology to make personalization emulation anatomical model |
Non-Patent Citations (1)
| Title |
|---|
| 肿瘤体外3D模型的研究进展;梁熠成等;《现代肿瘤医学》;20160401;第1559-1661页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108274734A (en) | 2018-07-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Peng et al. | 3D bioprinting for drug discovery and development in pharmaceutics | |
| US20220323649A1 (en) | Three dimensional microtissue bioprinter | |
| Funamoto et al. | A novel microfluidic platform for high-resolution imaging of a three-dimensional cell culture under a controlled hypoxic environment | |
| CN102586105B (en) | Microfluidic diffusion and open intervening cell culture array chip and fabrication method and application thereof | |
| Jagtiani et al. | In vitro blood brain barrier models: An overview | |
| CN112226363B (en) | Device and method for culturing high-flux organoid by utilizing microarray deep well | |
| CN108274734B (en) | System and method for manufacturing in-vitro 3D tumor microsphere model with controllable core-shell structure | |
| CN108327263A (en) | 3 D-printing device and method based on two-component hydrogel | |
| CA2995474C (en) | A microfluidic hydrodynamic shuttling chip device for highthroughput multiple single cells capture | |
| Puleo et al. | Applications of MEMS technologies in tissue engineering | |
| Ge et al. | Development of multi-dimensional cell co-culture via a novel microfluidic chip fabricated by DMD-based optical projection lithography | |
| Hou et al. | Multidimensional controllable fabrication of tumor spheroids based on a microfluidic device | |
| Zhang et al. | Recent progresses in novel in vitro models of primary neurons: A biomaterial perspective | |
| CN112430542A (en) | Integrated microfluidic three-dimensional tumor chip and bionic method | |
| Mohan et al. | TANDEM: biomicrofluidic systems with transverse and normal diffusional environments for multidirectional signaling | |
| CN103360459B (en) | Method of protein crystallization and open constant current diffusible proteins matter crystalline arrays chip of constant current diffusion and preparation method thereof and application | |
| Farshidfar et al. | The feasible application of microfluidic tissue/organ-on-a-chip as an impersonator of oral tissues and organs: a direction for future research | |
| CN109289947A (en) | Gel base micro-fluidic chip and its manufacturing method based on secondary cross-linking | |
| Yan et al. | Revolutionizing the female reproductive system research using microfluidic chip platform | |
| US20220395830A1 (en) | Array platform for three-dimensional cell culturing and drug testing and screening | |
| KR20200008688A (en) | 3 dimensional cell culture assay | |
| US11712696B2 (en) | Drug screening platform simulating hyperthermic intraperitoneal chemotherapy | |
| US20230151325A1 (en) | Method for screening for target cells or cells, and biological culture chip | |
| CN110076965A (en) | A kind of heat flow flux type multiple flow passages injection structure and its injection moulding process | |
| CN218561453U (en) | Mould of high-flux bionic in-vitro tumor drug sieve model |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |