CN107320779B - Method and device for preparing in-vitro three-dimensional tissue model - Google Patents
Method and device for preparing in-vitro three-dimensional tissue model Download PDFInfo
- Publication number
- CN107320779B CN107320779B CN201710481286.3A CN201710481286A CN107320779B CN 107320779 B CN107320779 B CN 107320779B CN 201710481286 A CN201710481286 A CN 201710481286A CN 107320779 B CN107320779 B CN 107320779B
- Authority
- CN
- China
- Prior art keywords
- layer
- vitro
- photocuring
- preparing
- tissue
- 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.)
- Expired - Fee Related
Links
- 238000013334 tissue model Methods 0.000 title claims abstract description 42
- 238000000338 in vitro Methods 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 23
- 210000004027 cell Anatomy 0.000 claims abstract description 48
- 238000000016 photochemical curing Methods 0.000 claims abstract description 48
- 210000001519 tissue Anatomy 0.000 claims abstract description 45
- 239000002131 composite material Substances 0.000 claims abstract description 37
- 239000000017 hydrogel Substances 0.000 claims abstract description 17
- 230000005684 electric field Effects 0.000 claims abstract description 12
- 238000005516 engineering process Methods 0.000 claims abstract description 12
- 239000003102 growth factor Substances 0.000 claims abstract description 10
- 238000001723 curing Methods 0.000 claims abstract description 8
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 claims abstract description 7
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 claims abstract description 7
- 210000002744 extracellular matrix Anatomy 0.000 claims abstract description 7
- 239000011159 matrix material Substances 0.000 claims abstract description 5
- 239000011664 nicotinic acid Substances 0.000 claims abstract description 4
- 230000033001 locomotion Effects 0.000 claims description 28
- 230000007246 mechanism Effects 0.000 claims description 16
- 102000008186 Collagen Human genes 0.000 claims description 10
- 108010035532 Collagen Proteins 0.000 claims description 10
- 229920001436 collagen Polymers 0.000 claims description 10
- 230000003287 optical effect Effects 0.000 claims description 8
- 239000002202 Polyethylene glycol Substances 0.000 claims description 7
- 229920001223 polyethylene glycol Polymers 0.000 claims description 7
- WOBHKFSMXKNTIM-UHFFFAOYSA-N Hydroxyethyl methacrylate Chemical compound CC(=C)C(=O)OCCO WOBHKFSMXKNTIM-UHFFFAOYSA-N 0.000 claims description 4
- 230000002572 peristaltic effect Effects 0.000 claims description 4
- XMLYCEVDHLAQEL-UHFFFAOYSA-N 2-hydroxy-2-methyl-1-phenylpropan-1-one Chemical compound CC(C)(O)C(=O)C1=CC=CC=C1 XMLYCEVDHLAQEL-UHFFFAOYSA-N 0.000 claims description 3
- 125000000954 2-hydroxyethyl group Chemical group [H]C([*])([H])C([H])([H])O[H] 0.000 claims description 3
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 claims description 3
- CQAIBOSCGCTHPV-UHFFFAOYSA-N bis(1-hydroxycyclohexa-2,4-dien-1-yl)methanone Chemical compound C1C=CC=CC1(O)C(=O)C1(O)CC=CC=C1 CQAIBOSCGCTHPV-UHFFFAOYSA-N 0.000 claims description 3
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 claims description 3
- 229920001817 Agar Polymers 0.000 claims description 2
- 239000008272 agar Substances 0.000 claims description 2
- 230000010261 cell growth Effects 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000007493 shaping process Methods 0.000 claims description 2
- 238000007639 printing Methods 0.000 abstract description 3
- 239000010410 layer Substances 0.000 description 39
- 239000000243 solution Substances 0.000 description 32
- 210000004872 soft tissue Anatomy 0.000 description 7
- 238000010276 construction Methods 0.000 description 6
- 239000011259 mixed solution Substances 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 230000002792 vascular Effects 0.000 description 6
- 230000007547 defect Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 210000004185 liver Anatomy 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000002356 single layer Substances 0.000 description 3
- 239000002407 tissue scaffold Substances 0.000 description 3
- 230000006378 damage Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000004720 dielectrophoresis Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- 230000002440 hepatic effect Effects 0.000 description 2
- 210000002570 interstitial cell Anatomy 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 210000003556 vascular endothelial cell Anatomy 0.000 description 2
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- -1 cells Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000010287 polarization Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 230000025366 tissue development Effects 0.000 description 1
- 230000009772 tissue formation Effects 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3633—Extracellular matrix [ECM]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Transplantation (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Dermatology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Botany (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Urology & Nephrology (AREA)
- Manufacturing & Machinery (AREA)
- Materials Engineering (AREA)
- Dispersion Chemistry (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
A method and a device for preparing an in-vitro three-dimensional tissue model comprise the following steps: (2) preparing a photocuring composite solution mixed with cells by using an extracellular matrix material containing the cells and growth factors, a photocuring hydrogel and a photoinitiator; (3) adding the prepared photocuring composite solution into a forming tray; a variable controllable electric field is generated by the control electrode array, and the cells are manipulated by means of dielectrophoretic force to move and reach a target area; (4) importing a layer of tissue pattern data into a Digital Micromirror Device (DMD) chip to generate a photomask of the layer of tissue pattern, and curing the photocuring composite solution by using a surface exposure technology; (5) and (5) after finishing the photocuring of one layer of tissue, repeating the steps (3) and (4), performing cell manipulation and photocuring of the next layer of tissue, and accumulating layer by layer to obtain the in-vitro three-dimensional bionic tissue model. The invention can solve the problems of difficult cell manipulation, low printing speed and the like in the prior art.
Description
Technical Field
The invention relates to the technical field of in-vitro tissue model preparation, in particular to a method and a device for preparing an in-vitro three-dimensional tissue model.
Background
Tissue, organ defects or dysfunction due to various causes are major causes of harm to human health, and repair and functional reconstruction of tissue and organ defects are challenges facing the medical field. The development of tissue engineering brings new treatment hope for the damage and the functional deletion of tissues and organs, applies engineering and biological principles, jointly or independently uses biological materials, cells, growth factors and the like, reconstructs tissue and organ models in vitro, simulates factors such as tissue development, local microenvironment, biomechanical stimulation and the like, and is of great help for the research of pathology and pharmacology.
In recent years, the 3D bioprinting technology has been applied more widely in the construction of in vitro tissue models, and can individually control cell distribution and precisely control cell formation. However, extrusion printing is frequently applied in tissue construction at present, the mode has high requirements on the mechanical property of materials, high precision is often difficult to realize, the point-by-point scanning mode is low in efficiency, and the method is particularly prominent in a repetitive structure. The surface exposure technology based on photocuring is favored by researchers due to the characteristics of high precision, high efficiency and strong applicability.
Chinese patent document 201410280450.0 relates to a method for manufacturing an artificial soft tissue with a vascular network flow channel, which comprises the steps of firstly designing a soft tissue stent model with a vascular structure, layering the models one by one at equal intervals, and manufacturing photomask plates of all layers; then uniformly mixing the cells with the collagen solution, and injecting photocuring hydrogel and a photoinitiator to obtain a photocuring composite solution; injecting the photocuring composite solution onto a workbench, covering a photomask plate, curing the photocuring composite solution by using a surface exposure technology, and then curing and accumulating layer by layer to obtain a photocuring hydrogel soft tissue scaffold with a vascular structure; and (3) planting vascular endothelial cells in the vascular structure of the stent, enabling the vascular endothelial cells to be attached to the surface of the vascular duct, and performing static culture and dynamic culture in vitro to obtain the artificial soft tissue body with the vascular network flow channel.
The method can solve the problems of survival of cells in the large tissue engineering soft tissue scaffold in the repair of large defect of the soft tissue and the problems of manufacturing and vascularization of the blood vessel network of the soft tissue scaffold, has strong applicability and can also provide certain precision. But the disadvantage is that the cells in the complex solution can not be manipulated, so that the cells can be controlled and reach the target position to form a complex structure. In the construction of an in vitro three-dimensional tissue model, in order to be more lifelike and bionic, certain cells need to be limited in a certain area so as to simulate the tissue structure and function to a certain extent, which is also a difficult problem to be solved in practical research.
Disclosure of Invention
The invention mainly aims to overcome the defects of the prior art and provides a method and a device for preparing an in-vitro three-dimensional tissue model so as to solve the problems that cells are difficult to manipulate, the printing speed is low and the like in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for preparing an in vitro three-dimensional tissue model comprises the following steps:
(2) preparing a photocuring composite solution mixed with cells by using an extracellular matrix material containing the cells and growth factors, a photocuring hydrogel and a photoinitiator;
(3) adding the prepared light-cured composite solution into a forming tray, wherein the addition amount of the composite solution is determined by the preset thickness of the layer of tissue; a variable controllable electric field is generated by the control electrode array, and the cells are manipulated by means of dielectrophoretic force to move and reach a target area;
(4) importing a layer of tissue pattern data into a Digital Micromirror Device (DMD) chip, generating a photomask of the layer of tissue pattern through each micro reflector, and curing the photocuring composite solution by using a surface exposure technology;
(5) and (5) after finishing the photocuring of one layer of tissue, repeating the steps (3) and (4), performing cell manipulation and photocuring of the next layer of tissue, and accumulating layer by layer to obtain the in-vitro three-dimensional bionic tissue model.
Further:
before the step (2), the following steps are also included: (1) the method comprises the steps of designing an in-vitro three-dimensional tissue model by using computer aided design software, layering the designed tissue model one by one, obtaining tissues of each layer after layering, and generating pattern data of each layer.
In the step (3), the solution dosage newly entering the composite solution is controlled by a precise peristaltic pump or an injector, so that the thickness of each layer is controlled, and preferably, the thickness of each layer is 20-100 μm.
In the step (2), the photocuring composite solution contains a plurality of cells and is matched with corresponding growth factors and extracellular matrix suitable for cell growth.
Wherein different cells can be used in different layers of the tissue according to the requirements of tissue construction, and are matched with growth factors and collagen which are suitable for the cells.
The extracellular matrix material in the step (2) is one or a mixture of collagen, hydrogel, agar and the like; the light-cured hydrogel is polyethylene glycol acrylate or polyethylene glycol methacrylate; the photoinitiator is 2-hydroxy-4' - (2-hydroxyethyl) -2-methyl p-hydroxybenzoate or 1-hydroxyphenyl ketone or 2, 2-dimethoxy-1, 2-diphenylmethane 1-1 or 2-hydroxy 2-methyl propiophenone; the mass concentration of the photocuring hydrogel in the photocuring composite solution is 10-30%, and the mass concentration of the photoinitiator is 0.1-1%.
The laser power density is 10-1000 mW/cm ^2 when the surface exposure technology is adopted to cure the photocuring composite solution in the step (4), and the laser wavelength is generally 350-400 nm; preferably, the exposure time is 20-30 s.
An apparatus for preparing an in vitro three-dimensional tissue model, comprising: the device comprises a support (101), a motion platform (102), a lower microelectrode array (103), an upper electrode plate (104), a light path device (106), a support upright post (108), a rotating device (107), a forming tray (109), a control unit (110), an X-direction motion mechanism (201), a Y-direction motion mechanism (202) and a lifting device (105); the lifting device (105) is mounted on the support (101) and coupled to the motion platform (102); the X-direction movement mechanism (201) and the Y-direction movement mechanism (202) are mounted on the movement platform (102), the movement platform (102) is mounted on the support (101), the lower microelectrode array (103) is further mounted on the movement platform (102), the control unit (110) is connected with the light path device (106) and the lower microelectrode array (103) to control the on-off of each microelectrode and a high level, and meanwhile, the upper electrode plate (104) is connected with a low level to form a controllable variable electric field; the shaping tray (109) is mounted above the lower microelectrode array (103); the supporting upright post (108) is fixedly connected with the bracket (101); the rotating device (107) is mounted on the supporting column (108) and can rotate around the supporting column (108), and the upper electrode plate (104) and the light path device (106) are mounted on the rotating device (107).
Further:
the potential difference between the high level and the low level of the controllable variable electric field is 10-20V.
The rotating device (107) is provided with 2 clamping devices which respectively correspond to the light path device (106) and the upper electrode plate (104) and are positioned right above the moving platform (102).
The optical path device (106) comprises a laser light source (303), a DMD chip (301) and a lens device (302), wherein laser is generated by the laser light source (303), is output from the optical path device (106) through the DMD chip (301) and the lens device (302), and irradiates the composite solution on the forming tray (109); the DMD chip (301) is controlled by the control unit (110) to form a pattern of a photomask by laser.
Compared with the prior art, the invention has the following advantages:
1. in the preparation method of the in-vitro three-dimensional tissue model, the adopted cell manipulation method and the photomask generation method have strong applicability, can be flexibly adjusted according to requirements, and can be used for preparing various tissue models.
2. The preparation method and the preparation device provided by the invention have low requirements on the mechanical properties of the materials, and the range of usable materials is wide.
3. The cells are manipulated by utilizing the dielectrophoresis principle, the cells can be moved to a target area, the polarization properties of different types of cells are different, and the dielectrophoresis forces applied to different electric fields are different, so that different cells can be manipulated to different areas by utilizing the point, the cell distribution areas of each layer of tissue are different, and a more complex three-dimensional tissue structure can be formed.
4. By adopting the photocuring forming method, the normal-temperature forming can be realized, and the survival rate of cells is high. The DMD chip is used for generating a photomask pattern, a mask used in the prior art is replaced, and a mask does not need to be manufactured for each layer of organizational structure, so that the material and time are saved, the efficiency is improved, and the method is convenient and quick.
5. The preparation device provided by the invention is simple in structure and convenient to operate. The motion platform can realize the motion of three degrees of freedom, and is convenient for positioning the organization structure. The rotating device enables the upper electrode plate and the light path device to be switched conveniently, the connection between the cell manipulation step and the light curing step is continuous and smooth, and the precision and the rapid forming of the tissue structure are facilitated.
Drawings
FIG. 1 is a schematic three-dimensional structure of an embodiment of an apparatus for preparing an in vitro three-dimensional tissue model according to the present invention.
Fig. 2 is a diagram showing the positional relationship between the moving platform and the tray.
Fig. 3 is a schematic diagram of the inside of the optical path apparatus.
FIG. 4 is a schematic flow chart of the single-layer tissue formation in the method for preparing the in-vitro three-dimensional tissue model according to the present invention.
FIG. 5 is a schematic flow chart of an apparatus for preparing an in vitro three-dimensional tissue model according to the present invention.
FIG. 6 is a schematic illustration of the use of varying electric fields to manipulate cells to a target location.
FIG. 7 is a schematic illustration of the construction of a single layer lobular tissue model.
Detailed Description
The embodiments of the present invention will be described in detail below. It should be emphasized that the following description is merely exemplary in nature and is not intended to limit the scope of the invention or its application.
Referring to fig. 1 to 3, in an embodiment, an apparatus for preparing an in vitro three-dimensional tissue model includes a support (101), a control unit (110), a motion platform (102), a lower microelectrode array (103), an upper electrode plate (104), an optical path device (106), a support pillar (108), and a rotation device (107), wherein: the device also comprises an X-direction movement mechanism (201), a Y-direction movement mechanism (202) and a lifting device (105); the lifting device (105) is arranged in the upright post of the bracket (101); the X-direction movement mechanism (201) and the Y-direction movement mechanism (202) are arranged on a movement platform (102), the movement platform (102) is arranged on a support (101), and a microelectrode array (103) is also arranged on the movement platform (102); the tray (109) is arranged above the microelectrode array (103); the bottom of the support upright post (108) is fixedly connected with the bottom of the bracket (101); the rotating device (107) is arranged on the supporting upright post (108), and the upper electrode plate (104) and the light path device (106) are fixedly connected with the rotating device (107).
The light path device (106) and the upper electrode plate (104) are arranged on a rotating device (107), and the rotating device (107) is arranged on a supporting upright post (108) and can rotate around the upright post; the rotating device (107) is provided with 2 clamping devices which respectively correspond to the positions of the light path device (106) and the upper electrode plate (104) right above the moving platform (102), and the switching between the upper electrode plate (104) and the light path device (106) can be realized in the process.
The microelectrode array (103) is arranged on the moving platform (102), the microelectrode array (103) is formed by arranging a plurality of tiny electrodes, the control unit (110) controls the tiny electrodes to be connected with a high level, meanwhile, the upper electrode plate (104) is connected with a low level to form a controllable variable electric field, and the potential difference between the high level and the low level is 10-20V.
As shown in fig. 3, the optical path device (106) includes a laser light source (303), a DMD chip (301) and a lens device (302), wherein laser light generated by the light source (303) is output from the optical path device (106) through the DMD chip (301) and the lens device (302) and is irradiated to the composite solution on the tray (109); the DMD chip (301) is controlled by a control unit (110) to form a pattern of a photomask by laser.
Referring to fig. 4 to 7, in another embodiment, a method for preparing an in vitro three-dimensional tissue model includes the following steps:
(1) the method comprises the steps of designing an in-vitro three-dimensional tissue model by using computer aided design software, layering the designed tissue model one by one, obtaining tissues of each layer after layering, and generating pattern data of each layer. The thickness of each layer can be selected within the range of 100 μm-1mm as required, and different thicknesses of each layer can be realized by different dosages of the compound solution.
(2) Uniformly mixing the cells, the growth factors and the collagen solution to obtain a mixed solution, then injecting photocuring hydrogel into the mixed solution, and then adding a photoinitiator to obtain the photocuring composite solution mixed with the cells. The cells can be used in different layers of tissues according to the requirements of tissue construction, and are matched with growth factors and collagen suitable for the cells. The light-cured hydrogel is polyethylene glycol acrylate or polyethylene glycol methacrylate; the photoinitiator is 2-hydroxy-4' - (2-hydroxyethyl) -2-methyl p-hydroxybenzoate or 1-hydroxyphenyl ketone or 2, 2-dimethoxy-1, 2-diphenylmethane 1-1 or 2-hydroxy 2-methyl propiophenone; the mass concentration of the photocuring hydrogel in the photocuring composite solution is 10-30%, and the mass concentration of the photoinitiator is 0.1-1%.
(3) The prepared photo-setting composite solution is injected into a sterile tray (109) on a table by using an external device such as a syringe or a peristaltic pump, and the amount of the composite solution to be added is determined by the thickness of the layer tissue. The upper electrode plate (104) is rotated to a position right above the moving platform (102), and the X-direction moving mechanism (201), the Y-direction moving mechanism (202) and the lifting device (105) are adjusted to enable the tray (109) to be in a proper position. The control unit (110) causes the microelectrode array (103) to generate a varying non-uniform electric field (602) to manipulate the cell (601) by dielectrophoretic forces to bring the cell (601) to a target region, as shown in fig. 6.
(4) Rotating the light path device (106) to the position right above the moving platform (102), guiding the layer of tissue pattern data into a Digital Micromirror Device (DMD) chip (301), reflecting incident light into or out of a lens through different rotation angles of each micro reflector to generate a photomask of the layer of tissue pattern, turning on a laser light source (303), and curing the photocuring composite solution by using a surface exposure technology. Wherein the laser power density is 10-1000 mW/cm ^2 and the laser wavelength is 350-400nm when the surface exposure technology is adopted to cure the photocuring composite solution. The exposure time is 20-30 s.
(5) And (5) after finishing the photocuring of one layer of tissue, repeating the steps (3) and (4), carrying out cell manipulation and photocuring of other layers of tissue, and accumulating layer by layer to obtain the in-vitro three-dimensional tissue model.
Example 1
The preparation method of the in vitro three-dimensional liver lobule tissue model comprises the following specific steps:
(1) according to the structural characteristics of the hepatic lobule tissue, an in-vitro three-dimensional hepatic lobule tissue model is designed by using computer aided design software, the designed tissue models are layered one by one, tissues of each layer are obtained after layering, and tissue pattern data of each layer are generated. The thickness of each layer was 100 μm.
(2) Uniformly mixing the cells, the growth factors and the collagen solution to obtain a mixed solution, then injecting photocuring hydrogel into the mixed solution, and then adding a photoinitiator to obtain the photocuring composite solution mixed with the cells. Wherein the cells are human liver cells (702), endothelial cells and interstitial cells (701), the collagen in the collagen solution is type I collagen, the mixing density of the cells in the mixed solution is 1 x [ (10) as the exclusion of 7/ml, and the concentration of the collagen in the mixed solution is 5 mg/ml; the photo-curing hydrogel is polyethylene glycol methacrylate; the photoinitiator is 2, 2-dimethoxy-1, 2-diphenylmethane 1-1; the mass concentration of the photocuring hydrogel in the photocuring composite solution is 30 percent, and the mass concentration of the photoinitiator is 1 percent.
(3) The prepared photo-curing composite solution is added into a forming tray (109) on a workbench by using an external device such as a precision peristaltic pump, and the amount of the added composite solution is calculated by the thickness of 100 mu m and the size of the forming tray. The upper electrode plate (103) is rotated to a position right above the moving platform (102), and the X-direction moving mechanism (201), the Y-direction moving mechanism (202) and the lifting device (105) are adjusted to enable the tray (109) to be in a proper position. The control unit (110) enables the microelectrode array (103) to generate variable uneven electric fields, and the cells are manipulated by means of dielectrophoretic force, so that the liver cells, the endothelial cells and the interstitial cells reach a target area as required.
(4) Rotating the light path device (106) to the position right above the moving platform (102), guiding the layer of tissue pattern data into a Digital Micromirror Device (DMD) chip (301), generating a photomask of the layer of tissue pattern, turning on a laser light source (303), and curing the layer of light-cured composite solution by using a surface exposure technology. Wherein the laser power is 300mW and the laser wavelength is 355nm when the surface exposure technology is adopted to cure the photocuring composite solution.
(5) After completion of the photocuring of one layer of tissue, a single-layer liver lobular tissue model was obtained, as shown in fig. 7. And (5) repeating the steps (3) and (4), performing cell manipulation and photocuring on other layers of tissues, and accumulating layer by layer to obtain the in-vitro three-dimensional liver lobule tissue model.
The foregoing is a more detailed description of the invention in connection with specific/preferred embodiments and is not intended to limit the practice of the invention to those descriptions. It will be apparent to those skilled in the art that various substitutions and modifications can be made to the described embodiments without departing from the spirit of the invention, and these substitutions and modifications should be considered to fall within the scope of the invention.
Claims (9)
1. A method for preparing an in vitro three-dimensional tissue model is characterized by comprising the following steps:
(1) designing an in-vitro three-dimensional tissue model by using computer aided design software, layering the designed tissue models one by one to obtain tissues of each layer, and generating pattern data of the tissues of each layer;
(2) preparing a photocuring composite solution mixed with cells by using an extracellular matrix material containing the cells and growth factors, a photocuring hydrogel and a photoinitiator;
(3) adding the prepared light-cured composite solution into a forming tray, wherein the addition amount of the composite solution is determined by the preset thickness of the layer of tissue; a variable controllable electric field is generated by the control electrode array, and the cells are manipulated by means of dielectrophoretic force to move and reach a target area;
(4) importing a layer of tissue pattern data into a digital micro-mirror device chip, generating a photomask of the layer of tissue pattern through each micro-reflector, and curing the photocuring composite solution by using a surface exposure technology;
(5) and (5) after finishing the photocuring of one layer of tissue, repeating the steps (3) and (4), performing cell manipulation and photocuring of the next layer of tissue, and accumulating layer by layer to obtain the in-vitro three-dimensional bionic tissue model.
2. The method of preparing an in vitro three-dimensional tissue model of claim 1, wherein: in the step (3), the dosage of the solution newly entering the composite solution is controlled by a precise peristaltic pump or an injector, so that the thickness of each layer is controlled, and the thickness of each layer is 20-100 mu m.
3. The method for preparing an in vitro three-dimensional tissue model according to any one of claims 1 to 2, wherein: in the step (2), the photocuring composite solution contains a plurality of cells and is matched with corresponding growth factors and extracellular matrix suitable for cell growth.
4. The method for preparing an in vitro three-dimensional tissue model according to any one of claims 1 to 2, wherein: the extracellular matrix material in the step (2) is one or a mixture of collagen, hydrogel and agar; the light-cured hydrogel is polyethylene glycol acrylate or polyethylene glycol methacrylate; the photoinitiator is 2-hydroxy-4' - (2-hydroxyethyl) -2-methyl p-hydroxybenzoate or 1-hydroxyphenyl ketone or 2, 2-dimethoxy-1, 2-diphenylmethane 1-1 or 2-hydroxy 2-methyl propiophenone; the mass concentration of the photocuring hydrogel in the photocuring composite solution is 10-30%, and the mass concentration of the photoinitiator is 0.1-1%.
5. The method for preparing an in vitro three-dimensional tissue model according to any one of claims 1 to 2, wherein: the laser power density of the photo-curing composite solution cured by adopting the surface exposure technology in the step (4) is 10-1000 mW/cm2The laser wavelength is 350-400nm, and the exposure time is 20-30 s.
6. An apparatus for preparing an in vitro three-dimensional tissue model, comprising: the device comprises a support (101), a motion platform (102), a lower microelectrode array (103), an upper electrode plate (104), a light path device (106), a support upright post (108), a rotating device (107), a forming tray (109), a control unit (110), an X-direction motion mechanism (201), a Y-direction motion mechanism (202) and a lifting device (105); the lifting device (105) is mounted on the support (101) and coupled to the motion platform (102); the X-direction movement mechanism (201) and the Y-direction movement mechanism (202) are mounted on the movement platform (102), the movement platform (102) is mounted on the support (101), the lower microelectrode array (103) is further mounted on the movement platform (102), the control unit (110) is connected with the light path device (106) and the lower microelectrode array (103) to control the on-off of each microelectrode and a high level, and meanwhile, the upper electrode plate (104) is connected with a low level to form a controllable variable electric field; the shaping tray (109) is mounted above the lower microelectrode array (103); the supporting upright post (108) is fixedly connected with the bracket (101); the rotating device (107) is mounted on the supporting column (108) and can rotate around the supporting column (108), and the upper electrode plate (104) and the light path device (106) are mounted on the rotating device (107).
7. The apparatus for preparing an in vitro three-dimensional tissue model according to claim 6, wherein: the potential difference between the high level and the low level of the controllable variable electric field is 10-20V.
8. An apparatus for preparing an in vitro three-dimensional tissue model according to claim 6 or 7, wherein: the rotating device (107) is provided with 2 clamping devices which respectively correspond to the light path device (106) and the upper electrode plate (104) and are positioned right above the moving platform (102).
9. An apparatus for preparing an in vitro three-dimensional tissue model according to any one of claims 6 to 7, wherein: the optical path device (106) comprises a laser light source (303), a DMD chip (301) and a lens device (302), wherein laser is generated by the laser light source (303), is output from the optical path device (106) through the DMD chip (301) and the lens device (302), and irradiates the composite solution on the forming tray (109); the DMD chip (301) is controlled by the control unit (110) to form a pattern of a photomask by laser.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710481286.3A CN107320779B (en) | 2017-06-22 | 2017-06-22 | Method and device for preparing in-vitro three-dimensional tissue model |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710481286.3A CN107320779B (en) | 2017-06-22 | 2017-06-22 | Method and device for preparing in-vitro three-dimensional tissue model |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107320779A CN107320779A (en) | 2017-11-07 |
| CN107320779B true CN107320779B (en) | 2020-03-27 |
Family
ID=60196079
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710481286.3A Expired - Fee Related CN107320779B (en) | 2017-06-22 | 2017-06-22 | Method and device for preparing in-vitro three-dimensional tissue model |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107320779B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108481734B (en) * | 2018-02-14 | 2019-12-03 | 北京大学 | 4D micro-nano Method of printing based on three-dimensional laser direct write |
| CN108578777B (en) * | 2018-05-06 | 2021-05-07 | 西北工业大学 | Preparation method of artificial hard bone scaffold with controllable concentration gradient of growth factor |
| CN110039773B (en) * | 2019-05-08 | 2020-07-28 | 清华大学 | Three-dimensional printer with composite process and printing method thereof |
| CN111249528B (en) * | 2020-01-20 | 2021-07-16 | 浙江大学 | Tissue engineering bone based on multilayer cell grid and preparation method thereof |
| CN111497224A (en) * | 2020-04-27 | 2020-08-07 | 深圳市呈仪科技有限公司 | 3D printing method of cell growth scaffold |
| CN113350574B (en) * | 2021-05-26 | 2022-11-18 | 泸州国之荣耀酒业有限公司 | Method for weaving patterned liver-like small She Weizu |
| CN113601834B (en) * | 2021-08-16 | 2023-06-23 | 杭州捷诺飞生物科技股份有限公司 | Three-dimensional forming method and system |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN201386022Y (en) * | 2009-05-08 | 2010-01-20 | 东南大学 | Low-dimensional nanostructure material assembled chip based on three-dimensional controllable dielectrophoresis |
| CN102866193A (en) * | 2012-09-04 | 2013-01-09 | 吴传勇 | Device and method for controlling particles in liquid based on dielectrophoresis |
| CN103057123A (en) * | 2013-01-23 | 2013-04-24 | 南通大学 | Three-dimensional organism printing system and method for preparing nerve regeneration implant based on three-dimensional organism printing system |
| CN103302860A (en) * | 2013-06-08 | 2013-09-18 | 王健犀 | Light-curing three-dimensional printer based on digital light processing (DLP) projection |
| CN103448249A (en) * | 2013-09-13 | 2013-12-18 | 张靖 | Surface molded 3D (Three Dimensional) printing method and system |
| CN103921444A (en) * | 2014-05-04 | 2014-07-16 | 中山市东方博达电子科技有限公司 | Photocuring 3D printer, and photocuring 3D printing method and device |
| CN104382670A (en) * | 2014-12-08 | 2015-03-04 | 西安交通大学 | Bionic construction method of artificial organics |
| WO2015038914A1 (en) * | 2013-09-12 | 2015-03-19 | The Board Of Regents Of The University Of Texas System | 3d printing with nucleic acid adhesives |
| CN104669625A (en) * | 2015-03-12 | 2015-06-03 | 上海联泰三维科技有限公司 | Photo-curing three-dimensional printing method and printing device based on projection |
| CN106799835A (en) * | 2017-02-10 | 2017-06-06 | 深圳摩方新材科技有限公司 | A kind of photocuring 3D printer based on dot matrix display |
-
2017
- 2017-06-22 CN CN201710481286.3A patent/CN107320779B/en not_active Expired - Fee Related
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN201386022Y (en) * | 2009-05-08 | 2010-01-20 | 东南大学 | Low-dimensional nanostructure material assembled chip based on three-dimensional controllable dielectrophoresis |
| CN102866193A (en) * | 2012-09-04 | 2013-01-09 | 吴传勇 | Device and method for controlling particles in liquid based on dielectrophoresis |
| CN103057123A (en) * | 2013-01-23 | 2013-04-24 | 南通大学 | Three-dimensional organism printing system and method for preparing nerve regeneration implant based on three-dimensional organism printing system |
| CN103302860A (en) * | 2013-06-08 | 2013-09-18 | 王健犀 | Light-curing three-dimensional printer based on digital light processing (DLP) projection |
| WO2015038914A1 (en) * | 2013-09-12 | 2015-03-19 | The Board Of Regents Of The University Of Texas System | 3d printing with nucleic acid adhesives |
| CN103448249A (en) * | 2013-09-13 | 2013-12-18 | 张靖 | Surface molded 3D (Three Dimensional) printing method and system |
| CN103921444A (en) * | 2014-05-04 | 2014-07-16 | 中山市东方博达电子科技有限公司 | Photocuring 3D printer, and photocuring 3D printing method and device |
| CN104382670A (en) * | 2014-12-08 | 2015-03-04 | 西安交通大学 | Bionic construction method of artificial organics |
| CN104669625A (en) * | 2015-03-12 | 2015-06-03 | 上海联泰三维科技有限公司 | Photo-curing three-dimensional printing method and printing device based on projection |
| CN106799835A (en) * | 2017-02-10 | 2017-06-06 | 深圳摩方新材科技有限公司 | A kind of photocuring 3D printer based on dot matrix display |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107320779A (en) | 2017-11-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107320779B (en) | Method and device for preparing in-vitro three-dimensional tissue model | |
| Ng et al. | Vat polymerization-based bioprinting—process, materials, applications and regulatory challenges | |
| Zhang et al. | Digital light processing based three-dimensional printing for medical applications | |
| Tan et al. | Applications of 3D bioprinting in tissue engineering: advantages, deficiencies, improvements, and future perspectives | |
| Bernal et al. | Volumetric bioprinting of complex living‐tissue constructs within seconds | |
| US11447744B2 (en) | Hypothermic 3D bioprinting of living tissues supported by perfusable vasculature | |
| Xue et al. | Digital light processing-based 3D printing of cell-seeding hydrogel scaffolds with regionally varied stiffness | |
| Warner et al. | Design and 3D printing of hydrogel scaffolds with fractal geometries | |
| Zhang et al. | Shedding light on 3D printing: Printing photo-crosslinkable constructs for tissue engineering | |
| Ovsianikov et al. | Laser fabrication of three-dimensional CAD scaffolds from photosensitive gelatin for applications in tissue engineering | |
| Bejoy et al. | An insight on advances and applications of 3d bioprinting: A review | |
| Bartolo et al. | 3D bioprinting: Materials, processes, and applications | |
| US12007687B2 (en) | Microfluidics-enabled multimaterial stereolithographic printing | |
| Mo et al. | Advances in digital light processing of hydrogels | |
| JP2022507083A (en) | Compositions and Methods for Printing Tertiary Structure Corresponding to Biomaterials | |
| Koo et al. | Laser-assisted biofabrication in tissue engineering and regenerative medicine | |
| Kumar et al. | 3D bioprinting of nature-inspired hydrogel inks based on synthetic polymers | |
| Huh et al. | Three-dimensional bioprinting for tissue engineering | |
| WO2021146466A1 (en) | Methods and systems for model generation | |
| Wang et al. | Digital micro-mirror device-based light curing technology and its biological applications | |
| Ge et al. | Bubble-based microrobots enable digital assembly of heterogeneous microtissue modules | |
| Zeußel et al. | Bio-inspired 3D micro structuring of a liver lobule via direct laser writing: A comparative study with SU-8 and SUEX | |
| Kiratitanaporn et al. | Multimodal three-dimensional printing for micro-modulation of scaffold stiffness through machine learning | |
| CN104328084A (en) | Rapid template-free cell graphing method | |
| US11528902B2 (en) | Method and apparatus for keeping artificially created tissues alive |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200327 |