CN113862152A - Modular plug-in for three-dimensional cell culture - Google Patents
Modular plug-in for three-dimensional cell culture Download PDFInfo
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- CN113862152A CN113862152A CN202111225575.XA CN202111225575A CN113862152A CN 113862152 A CN113862152 A CN 113862152A CN 202111225575 A CN202111225575 A CN 202111225575A CN 113862152 A CN113862152 A CN 113862152A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/12—Well or multiwell plates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5067—Liver cells
Abstract
The invention discloses a modular insert for three-dimensional cell culture. The modular plug-in unit designed by the invention takes a PMMA (polymethyl methacrylate) micro-pit array as a bottom surface, adopts a Transwell form, and can be used as a plug-in unit of a cell culture pore plate to be embedded into a cell culture pore for three-dimensional cell culture. Different from the traditional processing technologies with time and labor waste, such as photoetching, wet etching and the like, the bottom micro-pit array is obtained by processing through a numerical control milling machine. After the insert is subjected to low cell adhesion modification, the cell suspension is kept stand in the insert for 12h and then self-assembled to form cell spheres, and then three-dimensional culture is continued. The modular card design facilitates cell ball array transfer while allowing coupling to other devices. Meanwhile, the through structure on the side edge of the cantilever allows the cell balls in the plug-in unit to exchange substances with other cells at the bottom of the pore plate, and further the interaction between the cells is researched.
Description
Technical Field
The invention relates to the fields of multi-cell three-dimensional culture and the like, in particular to a modular plug-in unit for three-dimensional cell culture and provides a new means for researching the interaction between three-dimensional cells.
Background
Most cells in an organism exhibit a three-dimensional structure, with each cell interacting with neighboring cells and extracellular matrix (ECM) components. Cell/cell and cell/ECM interactions are key factors for morphogenesis, cell signaling, cell viability and functional maintenance. However, most conventional cell culture methods are based on 2D culture systems. Although they are well established, these 2D monolayer cultures lack tight 3D cell/cell interactions and therefore do not reflect the true in vivo environment, leading to a loss of tissue specificity and increasing uncertainty in their effectiveness. The methods for forming 3D cell aggregates commonly used by scholars at present mainly include a pendant drop method, a substrate group modification method, a low adhesion micro-pit method, a material wrapping method, and the like. Among them, the micropit method has the characteristics of easy operation and generalization, is distinct from many methods, and is widely used.
However, the traditional micro-pit processing mostly adopts processing means such as photoetching, etching and the like, and wastes time and labor. Meanwhile, the micro-pits are mostly integrated by direct chips or attached to the bottom of a pore plate, so that the flexibility is poor and the cell balls are inconvenient to transfer.
Interactions between cells (including organs) are an essential feature of multicellular organisms. Cell-cell interactions can arise through direct contact, such as interactions between different cells in a tissue; cell-cell interactions may also occur indirectly, such as through secretion interactions. The study of cell interactions (such as cancer development and migration, wound healing and stem cell development) has important implications for drug screening and tissue engineering, especially for the evaluation of systemic drugs. To establish a model for studying cell/cell interactions, co-culturing of two or more cell types is required. Conventional co-culture methods include adding different cell types directly to the same culture well or cell culture using a Transwell system. Although this method is simple, the model is based on a 2D monolayer of cells, which differ from the in vivo cells in terms of morphology, physiology, and gene expression. There is an urgent need for a tool that can efficiently perform three-dimensional cell interactions.
Disclosure of Invention
In order to solve the problems, the invention provides a modular plug-in unit for three-dimensional cell culture, and meanwhile, the study on the mutual group of cells can be carried out.
The design drawing and the object drawing are respectively shown in the figure 1A and the figure B. The method is characterized in that: the insert consists of an aperture plate cantilever (fig. 2A), a bottom micro-pit array (fig. 2B).
The orifice plate cantilever adopts an orifice plate cantilever structure the same as that of a Transwell, and comprises an upper end 2/3 circular-ring-shaped beam, a connecting arm for connecting the lower liquid pool wall and an annular liquid pool wall, and the orifice plate cantilever is compatible with a traditional cell culture orifice plate, so that a plug-in is hung on the orifice plate hole wall, and convenient assembly and taking are realized. And the liquid pool wall of the orifice plate cantilever is provided with a through structure, so that the cell balls in the plug-in unit and the cells at the bottom of the orifice plate can conveniently interact with each other through a culture medium (figure 3).
The bottom micro-pit array is positioned at the bottom of the plug-in unit and forms a liquid pool together with the annular liquid pool wall of the orifice plate cantilever. The micro-pit array is formed by arranging hundreds of hemispherical micro-pits in a close-packed manner and is used for generating cell spheres with high flux (figure 2).
Furthermore, the material of the orifice plate cantilever is polystyrene material or photosensitive resin.
Further, the orifice plate cantilever adopts injection molding or 3D printing processing.
Furthermore, the micro-pit array material at the bottom of the plug-in is methyl methacrylate.
Further, the micro-pit array at the bottom of the plug-in is processed by a milling machine.
Further, the diameter of the micro-pit is 100-800 microns. The modular insert of claim 1, wherein: the insert is compatible with conventional 6, 12 and 24-well cell culture plates.
The invention has the advantages that:
(1) compatible cell culture orifice plate, modular design, the cell ball of conveniently transferring.
(2) The method is stable, the batch difference is small, and the size of the small pit array is uniform.
(3) The processing method is simple, and complex operations such as photoetching, etching and the like are not needed.
(4) The study of the interaction between cells is convenient.
Drawings
In order to more clearly illustrate the technical scheme of the invention, the drawings used in the technical scheme description are simply introduced.
FIG. 1 is a schematic perspective view (A) and a schematic object view (B) of a modular insert of the present invention;
FIG. 2 is a layered block diagram of the modular insert of the present invention;
FIG. 3 is a schematic representation of a modular insert of the present invention for cell-cell interaction;
FIG. 4 is a photomicrograph of a cell spheronization microscope of a modular insert of the present invention;
Detailed Description
The present invention is further described with reference to the following specific examples, but the scope of the present invention is not limited by the examples, and if one skilled in the art makes some insubstantial modifications and adaptations to the present invention based on the above disclosure, the present invention still falls within the scope of the present invention. The PF127 related to the invention is Pluronic F127, which is a surfactant.
Example 1
High-throughput three-dimensional culture of hepatocytes and drug testing.
The used plug-in unit consists of a pore plate cantilever and a micro-pit array positioned at the bottom of the pore plate cantilever; the orifice plate cantilever comprises a cross beam with an upper end 2/3 in a ring shape, an annular liquid pool wall and a connecting arm for connecting the cross beam and the liquid pool wall, and the annular liquid pool wall is provided with a strip-shaped groove which is through inside and outside; the micro-pit array is positioned at the bottom of the plug-in unit and forms a liquid pool together with the annular liquid pool wall of the orifice plate cantilever.
And (3) processing polymethyl methacrylate by using a numerical control milling machine to obtain a micro-pit array at the bottom of the plug-in, printing the orifice plate cantilever by using a 3D printing technology, and sealing the orifice plate cantilever and the orifice plate cantilever. The insert is subjected to ultraviolet sterilization, then the insert is placed into a 6-well plate, the insert is soaked for 2 hours by using 0.2 percent PF127 to form a low-adhesion coating, then a cell suspension of human induced pluripotent liver cells is inoculated into the insert, the insert is kept stand for 24 hours, and the cells can self-assemble into cell spheres with the diameter of about 50-100 microns in a micro-pit with the diameter of 100 microns to form a three-dimensional culture state. Each insert can obtain about 400 hepatocyte spheroids, which can be used as high-throughput response individuals. Can be used for researching liver metabolism and hepatotoxicity of drugs, and can obtain fatty liver disease models by stimulating liver cells with short-chain fatty acids. A large number of hepatocyte spheroids with consistency are obtained by utilizing the plug-in micro-pit array, meanwhile, the plug-in form is convenient for hepatocyte transfer, downstream analysis is carried out, and the experimental flexibility is increased.
Example 2
The three-dimensional hepatocyte spheroids interact with lung cancer cells.
The used plug-in unit consists of a pore plate cantilever and a micro-pit array positioned at the bottom of the pore plate cantilever; the orifice plate cantilever comprises a cross beam with an upper end 2/3 in a ring shape, an annular liquid pool wall and a connecting arm for connecting the cross beam and the liquid pool wall, and the annular liquid pool wall is provided with a strip-shaped groove which is through inside and outside; the micro-pit array is positioned at the bottom of the plug-in unit and forms a liquid pool together with the annular liquid pool wall of the orifice plate cantilever.
And (3) processing polymethyl methacrylate by using a numerical control milling machine to obtain a micro-pit array at the bottom of the plug-in, printing the orifice plate cantilever by using a 3D printing technology, and sealing the orifice plate cantilever and the orifice plate cantilever. Then the insert is subjected to ultraviolet sterilization, and then the insert is placed into a 12-hole plate, and is soaked for 4 hours by using 0.2% PF127 to form a low-adhesion coating, then the cell suspension of the induced pluripotent liver cells is inoculated into the insert, and is kept stand for 12 hours, and the cells can self-assemble into 400-inch and 500-micron cell spheres in a micro-pit with the diameter of 800 microns to form a three-dimensional culture state. And then inoculating A549 lung cancer cells in other holes in the pore plate, transferring the plug-in unit into the corresponding hole after the cells adhere to the wall, and performing substance exchange and interaction on the two cells through a through structure on the plug-in unit. Applying lung cancer drugs such as gefitinib and the like to test the killing effect of the lung cancer drugs on lung cancer after liver metabolism. Further more lung cancer drugs can be tested, and liver metabolism, hepatotoxicity and lung cancer killing effect of the lung cancer drugs can be tested.
Claims (9)
1. A modular insert for three-dimensional cell culture, characterized by: the plug-in unit consists of a hole plate cantilever and a micro-pit array positioned at the bottom of the hole plate cantilever;
the orifice plate cantilever comprises a cross beam with an upper end 2/3 in a ring shape, an annular liquid pool wall and a connecting arm for connecting the cross beam and the liquid pool wall, and a strip-shaped groove is formed in the annular liquid pool wall;
the micro-pit array is positioned at the bottom of the plug-in unit and forms a liquid pool together with the annular liquid pool wall of the orifice plate cantilever, and the micro-pit array is formed by arranging hundreds of inwards-sunken hemispherical micro-pits in a close-packed mode.
2. A modular insert for three-dimensional cell culture according to claim 1, wherein: the orifice plate cantilever is made of polystyrene or photosensitive resin.
3. A modular insert for three-dimensional cell culture according to claim 1, wherein: the orifice plate cantilever adopts injection molding or 3D printing processing.
4. A modular insert for three-dimensional cell culture according to claim 1, wherein: the micro-pit array is made of polymethyl methacrylate.
5. A modular insert for three-dimensional cell culture according to claim 1, wherein: the micro-pit array is processed by a milling machine.
6. A modular insert for three-dimensional cell culture according to claim 1, wherein: the diameter of the hemispherical micro-pits of the micro-pit array is 100-800 microns.
7. A modular insert for three-dimensional cell culture according to claim 1, wherein: the insert is compatible with conventional 6, 12 and 24-well cell culture plates.
8. The modular insert for three-dimensional cell culture according to claim 1, wherein the strip-shaped groove formed in the wall of the annular liquid pool is a strip-shaped groove with a through inner part and a through outer part.
9. A method for three-dimensional cell culture and study of cell-cell interaction using the modular insert for three-dimensional cell culture according to any of claims 1-7, wherein:
(1) performing ultraviolet sterilization on the plug-in;
(2) placing the plug-in into the matched pore plate;
(3) soaking the plug-in piece for 2-4 h by using 0.2% PF 127;
(4) inoculating the cell suspension into the plug-in unit, standing for 12-24 h, and allowing the cells to self-assemble into cell balls, thus forming a three-dimensional culture state;
(5) another kind of cell is inoculated in other holes of the pore plate, and after the cell is attached to the wall, the plug-in unit is moved into the corresponding hole.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111225575.XA CN113862152A (en) | 2021-10-21 | 2021-10-21 | Modular plug-in for three-dimensional cell culture |
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| CN202111225575.XA CN113862152A (en) | 2021-10-21 | 2021-10-21 | Modular plug-in for three-dimensional cell culture |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154649A (en) * | 2020-02-28 | 2020-05-15 | 苏州大学 | Seal for manufacturing multi-concave gel sheet and using method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060172412A1 (en) * | 2005-02-02 | 2006-08-03 | Coletica | Support device for cells culture |
| CN111269830A (en) * | 2018-12-05 | 2020-06-12 | 中国科学院大连化学物理研究所 | Multi-organ chip based on microfluidic technology and application thereof |
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2021
- 2021-10-21 CN CN202111225575.XA patent/CN113862152A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060172412A1 (en) * | 2005-02-02 | 2006-08-03 | Coletica | Support device for cells culture |
| CN111269830A (en) * | 2018-12-05 | 2020-06-12 | 中国科学院大连化学物理研究所 | Multi-organ chip based on microfluidic technology and application thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154649A (en) * | 2020-02-28 | 2020-05-15 | 苏州大学 | Seal for manufacturing multi-concave gel sheet and using method |
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