CN209989412U - 3D high flux organ microchip - Google Patents
3D high flux organ microchip Download PDFInfo
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- CN209989412U CN209989412U CN201920426864.8U CN201920426864U CN209989412U CN 209989412 U CN209989412 U CN 209989412U CN 201920426864 U CN201920426864 U CN 201920426864U CN 209989412 U CN209989412 U CN 209989412U
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Abstract
The utility model discloses a 3D high flux organ microchip belongs to biological tissue engineering field. The chip comprises a liquid storage layer, a 3D culture layer and a bottom plate layer which are sequentially arranged in a layered manner. Utilize the double faced adhesive tape to accomplish the connection of stock solution layer, 3D cultivation layer and bottom plate layer, perhaps once only mould plastics and realize overall structure. 3D cell culture is carried out in culture micropores of the chip 3D culture layer, and a corresponding organ model is constructed in a bionic mode, so that the bionic capability is strong. The organ chip can be used for high-flux 3D drug screening and related research. Moreover, the liquid is convenient to operate, suitable for high flux and free of external equipment. The construction method is simple and effective.
Description
Technical Field
The utility model relates to a biological tissue engineering technical field, in particular to 3D high flux organ microchip.
Background
The research result shows that the three-dimensional cultured cells are more similar to the cells in vivo in various aspects such as cell morphology, gene and protein expression, cell metabolism and the like compared with the monolayer cells cultured in two dimensions. Current in vitro 3D cell culture techniques include growing multicellular spheroids in cell suspension, and growing cells embedded in an extracellular matrix. The 3D cell spheroid culture method formed by controlling the liquid drops and the low-adhesion cell culture plate has the advantages of simplicity and low cost. However, in this 3D spheroid cell culture technique, the physiological environment in the real body cannot be fully reflected. With the intensive research on the microenvironment of cells, extracellular matrix (ECM) plays an important role in maintaining physiological properties of cells, drug resistance, disease development, and cell metabolism. For example, during the process of tumor development and development, the 3D extracellular matrix significantly influences the response of tumor cells to microenvironment signals. There is therefore an urgent need to develop 3D cell culture methods within extracellular matrices in vitro. The drug screening platform based on 3D cell culture has more accurate results in the aspects of clinical drug screening and clinical drug testing, and can realize more accurate and efficient in-vitro drug response prediction compared with a 2D screening platform. The 3D drug screening test platform will become a bridge between the conventional 2D single-layer cell drug screening system and animal model screening. However, the existing 3D extracellular matrix culture platform has a single model and limited bionic capability, and the application cost is too high due to large consumption of cells, matrix materials and medicines, so that the 3D extracellular matrix culture platform is not widely popularized.
SUMMERY OF THE UTILITY MODEL
The embodiment of the utility model provides a 3D high flux organ microchip. The following presents a simplified summary in order to provide a basic understanding of some aspects of the disclosed embodiments. This summary is not an extensive overview and is intended to neither identify key/critical elements nor delineate the scope of such embodiments. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.
According to a first aspect of embodiments of the present invention, there is provided a 3D high-flux organ microchip, comprising, in order, a liquid storage layer, a 3D culture layer and a bottom plate layer, which are layered;
the liquid storage layer is provided with a plurality of liquid storage through holes, and the liquid storage through holes are used for storing culture liquid;
a 3D culture layer having a plurality of culture microwells for 3D cell culture;
the liquid storage through holes correspond to the culture micropores one by one.
In an alternative embodiment, the pore size of the reservoir through-hole is greater than or equal to the pore size of the culture microwell.
In an optional embodiment, the liquid storage through hole of the liquid storage layer is a liquid storage column hole.
In an alternative embodiment, a plurality of the liquid storage through holes form a through hole liquid storage area, and liquid containing grooves are formed on the liquid storage layer around the through hole liquid storage area.
In an optional embodiment, the liquid storage through hole of the liquid storage layer is a liquid storage column hole, and a frame is arranged on the liquid storage layer around the liquid storage area of the through hole in a surrounding manner to form a liquid containing groove; and the liquid storage column hole is positioned in the liquid containing groove.
In an alternative embodiment, the reservoir through-hole of the reservoir layer and/or the culture microwell of the 3D culture layer are straight wells.
In an alternative embodiment, the surface of the floor layer adjacent to the 3D culture layer 20 is hydrophobic.
In an alternative embodiment, an uneven roughened surface is formed on the surface of the floor layer corresponding to the culture wells of the 3D culture layer.
The embodiment of the utility model provides a technical scheme can include following beneficial effect:
the utility model discloses organ microchip can be used to high flux 3D drug screening. The method can bionically construct a 3D in-vitro organ model combined with high-throughput drug screening, and can be used for scientific research of related drugs and drug screening. The utility model discloses high flux organ microchip, liquid convenient operation, be fit for high flux operation and sign, do not need external equipment. The utility model discloses organ microchip preparation method is simple, and is effective, and bonding or integrated into one piece all can.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.
FIG. 1 is a schematic diagram of a 3D high flux organ microchip shown in accordance with an exemplary embodiment;
FIG. 2 is a schematic cross-sectional structure of the 3D high throughput organ microchip of FIG. 1;
FIG. 3 is a schematic cross-sectional structural view of a 3D high throughput organ microchip shown in accordance with an exemplary embodiment;
FIG. 4 is a schematic diagram illustrating a liquid storage layer configuration according to an exemplary embodiment;
FIG. 5 is a schematic diagram of a 3D high flux organ microchip shown in accordance with an exemplary embodiment;
FIG. 6 is a schematic cross-sectional structure of the 3D high throughput organ microchip of FIG. 5;
FIG. 7 is a schematic diagram of a 3D high flux organ microchip shown in accordance with an exemplary embodiment;
FIG. 8 is a schematic cross-sectional structure of the 3D high throughput organ microchip of FIG. 7;
FIG. 9 is a schematic diagram illustrating an exploded structure of a 3D high flux organ microchip, according to an exemplary embodiment;
FIG. 10 is a schematic diagram illustrating the construction of a floor layer according to an exemplary embodiment;
FIG. 11 is a schematic diagram illustrating the construction of a floor layer according to an exemplary embodiment;
FIG. 12 is a schematic diagram of a 3D high flux organ microchip shown in accordance with an exemplary embodiment;
FIG. 13 is a tumor cell photomicrograph of a 3D tumor model shown in accordance with an exemplary embodiment;
FIG. 14 is a tumor cell photomicrograph of a comparative 2D tumor model shown in accordance with an exemplary embodiment;
FIG. 15 is a tumor cell line proliferation characterization curve of a tumor model shown in accordance with an exemplary embodiment;
FIG. 16 is a 3D tumor model stromal contraction plot showing a seeded cell number of 2500, according to an exemplary embodiment;
FIG. 17 is a 3D tumor model stromal contraction plot with a seeded cell number of 5000, according to an exemplary embodiment;
FIG. 18 is a 3D tumor model stromal contraction plot with seeded cell number 10000, according to an example embodiment;
FIG. 19 is a graph showing the results of a 3D organ microchip tumor model and 2D plate for anti-tumor drug screening, according to an exemplary embodiment;
FIG. 20 is a graph of control and doxorubicin exposure fluorescence results for a 3D H460 tumor model shown in accordance with an exemplary embodiment;
description of reference numerals: 10. a liquid storage layer; 11. a liquid storage through hole; 110. a post (or, a cylindrical portion); 100. a groove; 101. a gap; 102. a frame; 1021. an outer sidewall; 103. a bottom surface; 20. a 3D culture layer; 21. culturing the micropores; 30. a floor layer; 31. rough surface.
Detailed Description
The technical solution of the present invention will be described clearly and completely below with reference to the embodiments of the present invention, and it should be understood that the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by a person skilled in the art without creative efforts belong to the protection scope of the present invention.
The 3D high-flux organ microchip according to the embodiment of the present invention is described with reference to fig. 1 to 12, and includes a liquid storage layer 10, a 3D culture layer 20, and a bottom plate layer 30, which are sequentially layered.
The liquid storage layer 10 is provided with a plurality of liquid storage through holes 11, and the liquid storage through holes 11 are used for storing culture liquid;
a 3D culture layer 20 having a plurality of culture micro-wells 21, the culture micro-wells 21 being used for 3D cell culture;
the liquid storage through holes 11 correspond to the culture micropores 21 one by one.
The utility model discloses organ microchip, can be used to high flux drug screening. The 3D high-flux organ microchip can be matched with commercial high-flux liquid transfer equipment and high-flux detection instruments (enzyme labeling instruments and high content imaging systems) through the design of a liquid storage through hole 11, cell planting, solution operation, result detection and the like. Various types of cells and matrixes (such as collagen and matrigel) are mixed and then planted in the culture micropores of the 3D high-flux organ microchip for 3D cell culture, different organ models are formed in a bionic mode, the bionic capacity is strong, and the method can be used for scientific research of related medicines and medicine screening. Such as tumor organ models, myocardial fibrosis organ models, liver injury organ models, and the like.
The utility model discloses the organ microchip is a 3D cell culture system, and it can realize the micro-consumption of cell, matrix material, reagent and medicine, solves the problem with high costs in the extensive drug screening process. Besides, the method can be applied to models with high function integration and strong bionic ability.
The utility model discloses the organ microchip, when using, the top layer can be regarded as to stock solution layer 10, sets up.
In the embodiment of the utility model, the liquid storage layer 10, the 3D culture layer 20 and the bottom plate layer 30 can be processed in layers, and the layers are sequentially stacked; or integrally formed.
When the liquid storage layer 10, the 3D culture layer 20 and the bottom plate layer 30 are processed in a layered manner, the layers are sequentially stacked, and adjacent two layers in the stacking relationship can have a connection relationship, such as adhesion; or in a lapped relationship, for example, three layers are sequentially stacked and then clamped and fixed by a clamp.
When the liquid storage layer 10, the 3D culture layer 20 and the bottom plate layer 30 are integrally formed, each layer may be formed in a layered manner by integral injection molding (e.g., integral injection molding with a mold), or laser etching. For example, a plurality of grooves are formed in one side surface of the plate body, the grooves are two layers of stepped grooves, the groove close to the surface of the plate body serves as a liquid storage through hole 11, and the other layer serves as a culture micropore 21, as shown in fig. 12.
In an alternative embodiment, the diameter of the liquid storage through hole 11 is larger than or equal to the diameter of the culture micro-hole 21.
In an alternative embodiment, the diameter of the liquid storage through hole 11 is 1-6 times the diameter of the culture micro-hole 21. Optionally, the pore diameter of the liquid storage through hole 11 is 2 to 3 times of the pore diameter of the culture micro-hole 21. Alternatively, the pore diameter of the reservoir through-hole 11 is 2.5 times the pore diameter of the culture micro-hole 21.
In an alternative embodiment, the liquid storage through hole 11 of the liquid storage layer 10 and/or the culture micro-holes 21 of the 3D culture layer 20 are straight holes, i.e. uniform in pore size. For example, when the cross section of the liquid storage through hole 11 is circular, the straight hole is a cylindrical hole. The straight hole has the advantages of simple focusing of a fluorescence microscope, simple screening and imaging of high content drugs and the like, and can also be a tapered hole with gradually changed depth.
In an alternative embodiment, the shape of the liquid storage through hole 11 of the liquid storage layer 10 and/or the culture micro-hole 21 of the 3D culture layer 20 may be a circular hole, a polygonal hole, or the like, or may be a hole with an irregular geometric shape. When the hole diameter is a round hole, the hole diameter is the diameter of the circle; when a polygonal aperture is used, the aperture is the diameter of the inscribed circle of the polygon.
In the embodiment of the utility model, stock solution layer 10 is used for storing culture solution such as cell culture medium or medicine diluent and to bionical microtissue transmission material, for whole system provides the necessary nutrient of growth or the medicine that needs the test. The structure and the arrangement of the liquid storage layer 10 and the liquid storage through hole 11 thereon are various, and the liquid storage through hole 11 can be formed to achieve the above purpose.
In an alternative embodiment, the liquid storage layer 10 may be made of polymethyl methacrylate (PMMA) or Polystyrene (PS), but is not limited to the listed materials, and may be implemented by laser perforation or one-time injection molding.
In an optional embodiment, the arrangement of the liquid storage through holes on the liquid storage layer 10 is compatible with commercial sample adding equipment and detectors (such as an elisa instrument, a high content imaging system and the like), and high-throughput sample adding and detection can be realized. E.g., 96-well, 384-well, etc.
In an alternative embodiment, the depth of the liquid storage through hole 11 of the liquid storage layer 10 is 1-10 mm.
Optionally, the depth of the liquid storage through hole 11 is 2-5 mm.
Optionally, the depth of the liquid storage through hole 11 is 3 mm.
In an alternative embodiment, the liquid storage through hole 11 of the liquid storage layer 10 has a hole diameter of 3-8 mm. Optionally, the hole diameter of the liquid storage through hole 11 of the liquid storage layer 10 is 6 mm.
In an alternative embodiment, the liquid storage layer 10 is a liquid storage plate, and a liquid storage through hole 11 is formed in the liquid storage plate, such as the liquid storage layer 10 shown in fig. 1 and 2. In this embodiment, the depth of the liquid storage through hole 11 is the thickness of the liquid storage plate 10.
In an alternative embodiment, the reservoir via 11 of the reservoir layer 10 is a reservoir hole (i.e., the reservoir via 11 has a cylindrical portion 110 with a convex surface). Specifically, the liquid storage column hole is formed by forming a through hole on a convex column (i.e., the column part 110) on the convex surface. At this time, the depth of the liquid storage column hole is the sum of the height of the column part 110 and the thickness of the base body of the liquid storage layer 10.
In this embodiment, when the liquid storage through hole 11 is in the form of a liquid storage column hole, the thickness of the substrate of the liquid storage layer 10 is not limited as long as it has sufficient strength to support the liquid storage column holes arranged thereon. The wall thickness of the projection 110 of the reservoir bore is also not limited.
In an alternative embodiment, the plurality of liquid storage through holes 11 constitute a through hole liquid storage region, and the liquid containing tank 100 is formed on the liquid storage layer around the through hole liquid storage region. The liquid container 100 contains a solution, such as sterile water or PBS buffer, for increasing the humidity of the entire well plate and reducing the evaporation degree.
Optionally, the liquid storage layer 10 is a liquid storage plate, and a liquid storage through hole 11 is formed in the liquid storage plate. The liquid containing groove 100 is a groove body which is arranged on the liquid storage plate around the liquid storage area of the through hole. Alternatively, as shown in fig. 3 and 4, the groove may be a groove that does not penetrate the upper and lower surfaces of the liquid storage plate 10. Of course, the liquid storage plate 10 may be perforated on the upper and lower surfaces thereof.
Alternatively, as shown in fig. 3 and 4, the liquid storage layer 10 has a liquid storage through hole 11 as a liquid storage column hole, and a frame 102 is provided around the liquid storage layer around the liquid storage area of the through hole to form a liquid containing tank 100. And, the liquid storage column hole 11 is in the liquid containing groove 100. In this embodiment, the liquid storage layer 10 is a grooved plate having liquid storage column holes 11. The depth of the liquid container 100 (corresponding to the height of the frame 102) is not limited, and may be greater than, less than or equal to the height of the column 110 of the liquid storage column hole 11. Alternatively, the depth of the liquid containing tank 100 is equal to the height of the column part 110 of the column hole 11, i.e., the upper end face of the column hole 11 is flush with the end face of the liquid containing tank 100.
Optionally, a gap 101 is provided between the through-hole liquid storage region and the inner side wall of the frame 102 (i.e., the inner circumferential wall of the liquid containing tank). Gap 101 is annular, and gap 101 can be used as a solution channel to provide a channel for PBS buffer to increase the humidity of the entire well plate and reduce the evaporation. Meanwhile, column hole gaps are formed among the liquid storage column holes 11 and can also be used as solution channels to provide channels for PBS (phosphate buffer solution) for increasing the humidity of the whole pore plate and reducing the evaporation degree.
In an alternative embodiment, the liquid storage layer 10 shown in fig. 5 and 6 is based on the structure of the liquid storage layer 10 shown in fig. 3 and 4, and the frame is a frame with a cross section in a shape like a Chinese character ji, one side of the frame is arranged on the liquid storage layer 10 as an inner side wall, and the lower edge of the other side of the frame is located outside the liquid storage layer 10 as an outer side wall. In this embodiment, the cross-section of the liquid storage layer 10 is in a shape of a nearly concave with an unsealed bottom surface.
Optionally, the outer sidewall 1021 of the frame 101 of the frame body with the cross-section of the "n" shape extends downward and beyond the bottom surface 103 of the liquid storage layer 10.
Optionally, the outer sidewall 1021 is stepped.
In an alternative embodiment, one side of the liquid storage layer 10 is covered with a membrane, and the other side is bonded with a double-sided adhesive tape. The diaphragm can be made of plastic film and is used for blocking dust and greasy dirt. The side to which the double-sided adhesive tape is bonded is the side to which the film layer 20 is attached, which increases the bonding strength between the two layers. And, it was confirmed that the double-sided adhesive layer did not generate toxicity to cells.
The embodiment of the utility model provides an in, 3D cultivates layer 20 for 3D cell culture micropore layer, has a plurality of cultivation micropores 21 on it (cultivation micropore 21 is the through-hole), can be used to cultivate the cell of mixing with three-dimensional matrix, more simulates in vivo cell growth microenvironment. Alternatively, the 3D culture layer 20 may be made of polymethyl methacrylate (PMMA) or Polystyrene (PS), but not limited to the listed materials, and may be formed by laser perforation or one-time injection molding.
In an alternative embodiment, the depth of the culture micro-holes of the 3D culture layer 20 is 0.5-2 mm. Optionally, the depth of the culture wells of the 3D culture layer 20 is 1 mm.
In an alternative embodiment, the aperture of the culture micro-holes 21 of the 3D culture layer 20 is 2-4 mm. Alternatively, the aperture of the culture micro-holes 21 of the 3D culture layer 20 is 2.5 mm.
In an alternative embodiment, the 3D culture layer 20 is a plate-shaped body, and a through hole is formed in the plate-shaped body, so as to obtain the 3D culture layer with a plurality of culture micropores. Alternatively, the 3D culture layer 20 is a PMMA plate.
The utility model discloses in the organ microchip of embodiment, bottom plate layer 30 plays the supporting role to and the cultivation micropore 21 of layer 20 is cultivateed to shutoff 3D. The bottom plate layer 30 may be made of glass, polymethyl methacrylate (PMMA) or Polystyrene (PS), but is not limited to these materials.
The thickness of the floor layer 30 cannot be too thick, e.g., 0.1 to 1mm, for the sake of microscopic observation and photographing. Such as 0.5 to 0.6 mm. E.g. 0.55 mm.
In an alternative embodiment, the surface of the bottom plate layer 30 adjacent to the 3D culture layer 20 is hydrophobic. It is favorable for culturing the substrate solution in the micropores 21 to be in a spherical shape.
Alternatively, an uneven rough surface is formed on the surface of the floor layer 30 corresponding to the culture micro-wells 21 of the 3D culture layer 20. Facilitating the attachment of the matrix solution. The form of the uneven rough surface is not limited.
Optionally, the rugged matte is a scratch pattern matte. The scratch pattern may be formed by scribing, e.g., using a laser. The scratch pattern is not limited, and may be a plurality of equidirectional scratches (as shown in fig. 10) or cross scratches (as shown in fig. 11).
Alternatively, the floor layer 30 is made of ground glass. The rough surface of the ground glass is utilized.
Optionally, the height difference between the concave part and the convex part on the rough surface is 0.1-0.3 mm. When the rough surface is a scratch pattern, the depth of the scratch is the height difference between the concave part and the convex part, and is 0.1-0.3 mm.
In an alternative embodiment, the liquid storage layer 10 and the 3D culture layer 20 are arranged in a sealed manner, and the liquid storage layer 20 is arranged between the bottom plate layer 30 and the 3D culture layer 20. The liquid storage layer 10, the 3D culture layer 20 and the bottom plate layer 30 are bonded by double-sided adhesive tape.
The utility model discloses 3D high flux organ microchip can utilize the double faced adhesive tape to bond the preparation with the three-layer. The method comprises the following steps:
s11, respectively cleaning the liquid storage layer 10, the 3D culture layer 20 and the bottom plate layer 30, and then drying;
s12, stacking and bonding the liquid storage layer 10, the 3D culture layer 20 and the bottom plate layer 30 in sequence by using double-sided adhesive tapes;
the preparation of the organ microchip was completed to obtain a 3D high-throughput organ microchip.
In the preparation method of this example, the 3D high-throughput organ microchip is prepared as three layers, including the liquid storage layer 10, the thin film layer 20 and the 3D culture layer 20, which are sequentially laminated and connected (bonded). Wherein, the liquid storage layer 10, the thin film layer 20 and the 3D culture layer 20 adopt the technical scheme described in the 3D high-flux organ microchip.
In this embodiment, optionally, in step S11, the liquid storage layer 10, the thin film layer 20, and the 3D culture layer 20 are washed with ethanol, then washed with deionized water, and then dried. Optionally, the drying temperature is from 45 ℃ to 65 ℃, optionally, 50 ℃.
Alternatively, the ethanol is analytically pure ethanol.
In this embodiment, optionally, in step S11, the liquid storage layer 10 is a liquid storage layer with a first double-sided adhesive disposed (attached or integrally processed) on one side, the 3D culture layer 20 is a 3D culture layer 20 with a second double-sided adhesive disposed (attached or integrally processed) on one side, and after the cleaning with ethanol, the liquid storage layer 10 and the 3D culture layer 20 are immersed in pure water for 12-24 hours.
In an alternative embodiment, the 3D high-throughput organ microchip is integrally formed. The 3D high-flux organ microchip is prepared by integral molding. For example, the organ microchip is obtained by opening a mold and integral injection molding. The mold is formed according to the structure of the organ microchip. Further, for example, laser etching is used to integrally form the organ microchip.
The utility model discloses 3D high flux organ microchip can be used to the organ models such as tumour organ model, myocardial fibrosis organ model and liver injury organ model's construction application.
The embodiment of the utility model provides an in, 3D high flux organ microchip constructs the external organ model that combines together with high flux drug screening, can be used to in the scientific research and the drug screening of relevant medicine. Of course, the organ models that can be constructed are not limited to the three types described above.
Utilize the utility model discloses method of 3D high flux organ microchip construction organ model of embodiment, including following step:
s31, sterilizing the 3D high-flux organ microchip, adding a mixed cell suspension containing model cells and a matrix material into the culture micropores 21 of the 3D culture layer 20, and culturing at 37 ℃ to form gel; obtaining a gel-formed organ microchip;
s32, adding a culture medium into the liquid storage through hole 11 of the liquid storage layer 10 of the gelatinized organ microchip, and culturing at 37 ℃ to complete the construction of the organ model.
The utility model discloses the organ model who founds has high flux screening performance, is a high flux bionical organ model, can be used to high flux drug screening.
In step 31, the mixed cell suspension containing the model cells and the matrix material is a single cell suspension comprising the matrix material and the model cells, and the pH value is 6.5-7.5. Wherein, the model cell refers to a cell corresponding to the constructed model, for example, when the constructed model is a tumor model, the model cell is HCT116 of Colorectal cancer (Colorectal), NCI-H460 and NCI-H226 of lung cancer (lung), MCF-7 of Breast cancer (Breast), MDA-MB-231 of Breast cancer (triple negative), and DU145 of Prostate cancer (Prostate). When the constructed organ model is a myocardial fibrosis model, the model cell is a myocardial cell line or human primary myocardial cell induced by human ipsc; when the constructed model is a liver injury model, the model cell is a hepatic cell line or a human primary hepatic cell induced by human ipsc.
Optionally, the pH value of the mixed cell suspension is 6.8-7.2.
Optionally, the pH of the matrix material mixed cell suspension is controlled by controlling the volume ratio of the single cell suspension, the alkaline solution and the matrix material. For example, the volume ratio of the single cell suspension to the matrix material is 1.5-4: 1, and an appropriate amount of alkaline solution is added to make the pH value of the matrix material mixed cell suspension neutral.
Alternatively, the ratio of the volume of single cell suspension to matrix material is 3.5: 1.5.
Alternatively, the alkaline solution can be NaOH or NaHCO3NaOH and NaHCO3One or more of the above.
Optionally, in step S31, the matrix material includes, but is not limited to, collagen or matrigel.
Alternatively, the concentration of collagen or matrigel is 5 mg/mL.
Alternatively, the mixed cell suspension is obtained by:
the centrifugation model cells are digested and the in situ tissue or cell line (2D cultured) is resuspended as a single cell suspension (e.g., 1.41X 10) with 0.25 (vt.)% pancreatin6cell/mL));
and mixing the single cell suspension with the matrix material, and adjusting the pH value to 6.5-7.5 to obtain the mixed cell suspension.
Alternatively, in step S31, the volume of the mixed cell suspension to be added to the culture wells 21 of the 3D culture layer 20 is not limited, and the seeding volume may be determined according to the concentration of the mixed cell suspension and the amount of seeding per well.
Optionally, the inoculation amount of each culture micropore 21 is 1000-10000.
Optionally, the volume of the mixed cell suspension is 5-10 μ L. The inoculation amount of the mixed cell suspension is microliter grade, and the dosage is small.
Optionally, in step 31, the incubation is at 37 ℃ to ensure gelling. The culture time is not limited, and the gelatinization is ensured. Optionally, culturing at 37 deg.C for 5-15 min, optionally 10 min.
Alternatively, in step S32, when the liquid storage layer 10 of the organ microchip has a liquid containing tank 100, further comprising adding PBS buffer or pure water into the liquid containing tank 100 of the liquid storage layer 10.
The following is a specific structure of the 3D high flux organ microchip, but the invention is not limited to the following embodiments of several structures.
Example 13D high throughput organ microchip I
As shown in FIGS. 1 to 2, the 3D high-throughput organ microchip I comprises a liquid storage layer 10, a 3D culture layer 20 and a substrate layer 30 which are connected in a laminated manner in this order.
Wherein, the liquid storage layer 10 is a liquid storage plate with holes (such as PMMA plate), and the liquid storage through hole 11 is a through hole arranged on the liquid storage plate body. The liquid storage through hole 11 is a circular straight hole (cylindrical hole), the aperture is 6mm, and the depth is 3 mm.
The 3D culture layer 20 is a culture plate with holes (such as PMMA plate), and the culture micropores 21 are through holes formed in the culture plate body. The culture micropores 21 are circular straight holes (cylindrical holes), the aperture is 2.5mm, and the depth is 1 mm.
The bottom plate layer 30 is made of PMMA or glass sheet, has a hydrophobic surface and has a thickness of 0.8 mm.
The 3D culture layer 20 is connected to the bottom surface of the liquid storage layer 10; the hydrophobic surface of the bottom plate layer 30 is attached on the bottom surface of the 3D culture layer 20. The liquid storage through holes 11 on the liquid storage layer 10 correspond to the culture micropores 21 on the 3D culture layer 20 one by one.
Example 2
As shown in FIGS. 3 and 4, the 3D high-throughput organ microchip II comprises a liquid storage layer 10, a 3D culture layer 20 and a substrate layer 30 which are sequentially laminated and connected.
Wherein, the liquid storage layer 10 is a liquid storage plate with holes (such as PMMA plate), and the liquid storage through hole 11 is a through hole arranged on the liquid storage plate body. A trough body, namely a liquid containing trough 100, is arranged on the liquid storage plate around the through hole liquid storage area of the liquid storage layer 10. The liquid containing groove 100 is annularly surrounded on the periphery of the liquid storage area of the through hole. The liquid storage through hole 11 is a circular straight hole (cylindrical hole), the aperture is 6mm, and the depth is 3 mm.
The 3D culture layer 20 is a culture plate with holes (such as PMMA plate), and the culture micropores 21 are through holes formed in the culture plate body. The culture micropores 21 are circular straight holes (cylindrical holes), the aperture is 2.5mm, and the depth is 1 mm.
The bottom plate layer 30 is made of PMMA or glass sheet, has a hydrophobic surface and has a thickness of 0.55 mm.
The 3D culture layer 20 is connected to the bottom surface of the liquid storage layer 10; the hydrophobic surface of the bottom plate layer 30 is attached on the bottom surface of the 3D culture layer 20. The liquid storage through holes 11 on the liquid storage layer 10 correspond to the culture micropores 21 on the 3D culture layer 20 one by one.
Example 33D high throughput organ microchip III
As shown in FIGS. 5 to 6, the 3D high-throughput organ microchip III comprises a liquid storage layer 10, a 3D culture layer 20 and a substrate layer 30 which are connected in sequence in a layer-by-layer manner.
The surface of the liquid storage layer 10 has a plurality of convex pillars (i.e. the pillar 110) with convex surfaces, and the convex pillars are provided with through holes to form a plurality of liquid storage pillar holes 11. A frame 102 is arranged around the liquid storage layer around the through-hole liquid storage area (formed by a plurality of liquid storage column holes 11) to form a liquid containing tank 100. And, the liquid storage column hole 11 is in the liquid containing groove 100. That is, the liquid storage layer 10 is a grooved plate (e.g., made of PMMA) with liquid storage column holes. Wherein, a gap 101 is arranged between the liquid storage area of the through hole and the inner side wall of the frame 102 (i.e. the inner peripheral wall of the liquid containing tank 100). The liquid storage column hole 11 is a circular straight hole (cylindrical hole), the aperture is 6mm, and the depth is 6 mm.
The 3D culture layer 20 is a culture plate with holes (such as PMMA plate), and the culture micropores 21 are through holes formed in the culture plate body. The culture micropores 21 are circular straight holes (cylindrical holes), the aperture is 2.5mm, and the depth is 1 mm.
The bottom plate layer 30 is made of PMMA or glass sheet, has a hydrophobic surface and has a thickness of 0.55 mm.
The 3D culture layer 20 is connected to the bottom surface of the liquid storage layer 10; the hydrophobic surface of the bottom plate layer 30 is attached on the bottom surface of the 3D culture layer 20. The column holes 11 on the liquid storage layer 10 correspond to the culture micropores 21 on the 3D culture layer 20 one by one.
Example 43D high throughput organ microchip IV
As shown in FIGS. 7 to 9, the 3D high-throughput organ microchip IV comprises a liquid storage layer 10, a 3D culture layer 20 and a substrate layer 30 which are connected in a laminated manner in this order.
In the liquid storage layer 10 of embodiment 2, the frame 102 is a frame with a cross section in a shape like a Chinese character ji, and the outer side wall 1021 of the frame 102 extends downward and exceeds the bottom surface 103 of the liquid storage layer 10. Namely, the cross section of the groove plate is in a shape of a nearly concave with an unclosed bottom surface.
The 3D culture layer 20 is a culture plate with holes (such as PMMA plate), and the culture micropores 21 are through holes formed in the culture plate body. The culture micropores 21 are circular holes with the aperture of 2.5mm and the depth of 1 mm.
The bottom plate layer 30 is made of PMMA or glass sheet, has a hydrophobic surface and has a thickness of 0.55 mm.
The 3D culture layer 20 is connected on the bottom surface 103 of the through hole liquid storage area of the liquid storage layer 10; the hydrophobic surface of the bottom plate layer 30 is attached on the bottom surface of the 3D culture layer 20. The column holes 11 on the liquid storage layer 10 correspond to the culture micropores 21 on the 3D culture layer 20 one by one.
Example 53D high throughput organ microchip
In the 3D high-flux organ microchip of example 4, on the basis of the three-layer structure organ microchips of examples 1 to 4, the surface of the bottom plate layer 30 adjacent to the 3D culture layer 20 has hydrophobicity, i.e., a hydrophobic surface, and the surface of the bottom plate layer 30 corresponding to the culture wells 21 of the 3D culture layer 20 has an uneven rough surface. The height difference between the concave part and the convex part on the rough surface is 0.1-0.3 mm. As shown in fig. 10, a scratch pattern rough surface is formed by scribing on the hydrophobic surface of the back sheet layer 30 corresponding to the culture micro-wells 21 of the 3D culture layer 20.
That is, the substrate layer 30 as described in fig. 10 is applied to the 3D high-flux organ microchip of fig. 1 and 2, and fig. 3 and 4, respectively, instead of the substrate layer 30 therein, to obtain 3D high-flux organ microchips i ', ii', iii ', and iv', respectively.
In the 3D high-throughput organ microchip according to examples 1 to 5, the pore diameters and depths of the reservoir through hole 11 and the culture micro-hole 21, the thickness of the bottom plate layer 30, and the like are not limited to the specific values given in the examples, and may be adjusted according to the actual situation. The shape of the holes of the liquid storage through hole 11 and the culture micropores 21 is not limited to a circular hole, and a straight hole is ensured.
The utility model discloses an in embodiment 1 to embodiment 5's 3D high flux organ microchip, stock solution layer 10 and 3D cultivate layer 20 and pass through double faced adhesive tape bonding connection. And, it was confirmed that the double-sided adhesive layer did not generate toxicity to cells.
In examples 1 to 5, the liquid storage layer 10, the 3D culture layer 20, or the bottom plate layer 30 may be integrally formed, for example, by injection molding or compression molding; or may be post-processed, such as by laser etching. The specific formation method is not limited as long as the structure is provided.
Example 6
The 3D high flux organ microchip of this example 6 is integrally formed. For example, the liquid storage column hole 11 and the culture micro-hole 21, and the liquid container 100 are formed on one plate body by means of integral injection molding or laser etching.
As shown in fig. 12, a plurality of convex pillars are formed on one side surface of a plate body, and two layers of stepped slot holes are dug on the convex pillars, the upper layer of stepped slot is used as a liquid storage through hole 11, and the lower layer of stepped slot is used as a culture micropore 21.
Wherein, the 3D high-flux organ microchips of the embodiments 1 to 5 can be obtained by integral molding.
Example 7
This example 7 provides a 3D high-throughput organ microchip preparation method, including the following steps:
s41, the liquid storage layer 10 is a liquid storage layer with one side surface adhered with first double-sided adhesive, the 3D culture layer 20 is a 3D culture layer 20 with one side surface adhered with second double-sided adhesive, and the liquid storage layer 10 and the 3D culture layer 20 are immersed in pure water for overnight, such as 12-24 hours.
And then respectively cleaning the liquid storage layer 10, the 3D culture layer 20 and the bottom plate layer 30 after the soaking pretreatment by using ethanol, washing by using deionized water, and drying at 50 ℃.
S42, bonding the side surface of the liquid storage layer 10 with the first double-sided adhesive to the side surface of the 3D culture layer 20 without the second double-sided adhesive; attaching a bottom plate layer 30 to the side of the 3D culture layer 20 having the second double-sided adhesive; and (5) pressing and bonding to finish the preparation of the organ microchip so as to obtain the 3D high-flux organ microchip.
In this example 7, the three-layer structured 3D high-throughput organ microchip of examples 1 to 5 was obtained according to the preparation method of this example 7 by using the liquid storage layer 10, the 3D culture layer 20 and the substrate layer 30 described in examples 1 to 5 as the liquid storage layer 10, the 3D culture layer 20 and the substrate layer 30 in step S41, respectively.
Example 83D construction of high throughput tumor organ models
This embodiment 8 is a method for constructing a tumor organ model, comprising the steps of:
s61, digesting the 2D cultured tumor cell line in the growth phase into single cell suspension by using 0.25(vt.) percent of pancreatin, centrifuging, resuspending, and resuspending into a density of 1.41 multiplied by 10 according to requirements6cell/mL of single cell suspension. Adding collagen (or other matrix materials such as matrigel with the concentration of 5mg/mL) and the single-cell suspension into a 1.5mL EP tube according to the volume ratio of the single-cell suspension to the matrix materials of 3.5: 1.5, and blowing and stirring the mixture uniformly by using a pipette to obtain mixed cell suspension, wherein the concentration of the collagen (or other matrix materials such as matrigel) is 5 mg/mL; the culture micro-wells 21 of the 3D culture layer 20 are inoculated by fast high-throughput transfer with a pipetting gun, and the amount of inoculation is determined according to the size of the micro-wells, for example, 5-10 μ L is inoculated in each culture micro-well with the aperture of 2.5 mm. After inoculation, the organ microchip is placed in a cell incubator and cultured for 10min at 37 ℃ to ensure that collagen can be well gelatinized, and the collagen is taken out to obtain the gelatinized organ microchip.
S62, adding a culture medium into the liquid storage through hole 11 of the liquid storage layer 10 of the gelatinized organ microchip, adding a PBS buffer solution into the liquid containing groove 100 of the liquid storage layer 10, and culturing at 37 ℃ to obtain a tumor model; and finishing the construction of the tumor model. The amount of the culture medium to be added to the reservoir through-hole 11 may be determined according to the size of the reservoir through-hole 11, for example, 100. mu.L of the culture medium may be added to the reservoir through-hole 11 having a 6mm aperture.
In this example 8, all operations involving the use of collagen are performed on an ice bin or other refrigeration equipment.
Next, the 3D high-throughput organ microchip IV of example 4 prepared by the preparation method of example 7 was used, and a tumor model IV was constructed by using example 8. Different 3D tumor models (abbreviated as 3D) were obtained by culturing with different tumor cell lines. The tumor cell lines in the growth phase cultured in 2D and the correspondingly constructed tumor models are shown in table 1 below.
TABLE 1
As a comparison, a comparative 2D cell planting and culture was designed: as a control group, the conventional 96-well plate was seeded with the same number and type of tumor cell lines at a density of 1.41X 106individual/mL of cell suspension, according to cell suspension: the volume ratio of the 1640 culture medium is 3.5:96.5, cell suspension with required volume and the 1640 culture medium are mixed, a pipetting gun is used for blowing and beating the mixture evenly and evenly to divide the mixture into 8 parts, the mixture is transferred and inoculated in a 96-well plate in a fast high-throughput manner by using a pipetting gun, and the number of 2D cells is ensured to be 5000 per well. The 96-well plate with the cells planted therein was placed in a 37 ℃ cell incubator and cultured. Based on the 7 tumor cell lines in Table 1 above, comparative tumor models 1-7 (2D-1-7) were obtained.
The following characterization tests were performed for the above 3D-IV-1 to 7 and 2D-1 to 7:
1. FIG. 13 is a 40-fold magnification photomicrograph of the tumor cells of 3D-IV-1, in which the tumor cells of 3D-IV-1 were cultured at 37 ℃ for 3 days in step S62. FIG. 14 is a photomicrograph of tumor cells in 2D-1 at 40X magnification, in which the tumor cells in 2D-1 were cultured in a cell culture chamber at 37 ℃ for 3 days. Comparing fig. 13 and 14, it can be seen that cells grew as monolayers in 2D plates, but more stereoscopically in 3D matrices, interacting with extracellular matrix and other cells.
2. 3D-IV-3 and 3D-IV-6 tumor models cultured with 3D high-flux organ microchip IV and corresponding 2D-3 and 2D-6 comparative tumor models cultured with 96-well plates, using cell titer blue to characterize comparative curves for tumor cell line proliferation, as shown in FIG. 15, wherein the curve indicated by "- ●" -is for the 3D-IV-6 tumor model and the curve indicated by "- ■" -is for the 2D-6 comparative tumor model, the curve indicated by "-is for the 3D-IV-3 tumor model and the curve indicated by" - "is for the 2D-3 comparative tumor model, it is clear from FIG. 15 that 3D-grown cells proliferated slower and more closely approached in vivo growth compared to 2D-cultured cells, and that 2D and 3D cell proliferation rates decreased with increasing cell number.
3. For MCF-7 cell line, 3D high throughput organ microchip IV on different number of cells, then using the microscope observation of 3D collagen contraction phenomenon. FIG. 16 is a 10-fold enlarged picture of 3D-IV-3 'with the cell number 2500, FIG. 17 is a 10-fold enlarged picture of 3D-IV-3 with the cell number 5000, and FIG. 18 is a 10-fold enlarged picture of 3D-IV-3' with the cell number 10000. Comparing fig. 16 to fig. 18, it can be seen that the constructed tumor model has a matrix shrinkage phenomenon during the growth process, and the matrix shrinkage phenomenon is more obvious as the number of the planted cells increases, and the matrix shrinkage phenomenon can be used as an evaluation index for drug screening.
4. Drug sensitive outcome detection
Screening the anti-tumor drugs: and (3) removing all culture mediums in 3D-IV-1-7 and 2D-1-7 which are constructed and cultured for 24 hours, adding 100 mu L of cell culture medium containing 10 mu M of anticancer drugs, and continuously culturing for 48 hours. Each 3D organ microchip and 2D 96-well plate (control) required the setting of a blank, negative control and positive control, each set with 6 secondary wells. Among the 18 anticancer drugs for differential screening of the 2D model and the 3D model are doxorubicin hydrochloride 1, epirubicin 2, mitomycin 3, 45-fluorouracil, gemcitabine 5, irinotecan 6 hydrochloride, paclitaxel 7, vorinostat 8, vincristine 9, etoposide 10, vinorelbine 11, sunitinib 12, temozolomide 13, lomustine 14, tegafur 15, cyclophosphamide 16, cisplatin 17, and capecitabine 18, but not limited to these anticancer drugs.
And (3) detecting a drug sensitivity result: the utility model discloses 3D organ microchip medicine screening system can use current quick detection means of medicine to characterize, and cell metabolic capacity evaluation system if add cell titer blue, compares cell metabolic capacity, evaluates the effect of drug action. After 48h drug stimulation, the media with drug was removed and cell titerblue stock solution was added: complete medium 1:5 (volume ratio) mixture, incubation at 37 ℃ for 1.5h, detection wavelength: 560em/590 exnm. Celltiter glo and steady glo evaluated 3D cultured cells for ATP and fluorescein. The high content imaging technology is used for imaging and characterizing the number of living and dead cells. The present embodiment is applicable to, but not limited to, the above characterization methods.
Wherein 3D-IV-1 (H460) is taken as a 3D tumor model, 2D-1(H460) is taken as a 2D tumor model, and the result of the calculation of the cell inhibition rate aiming at the 18 anti-cancer drugs is shown in figure 19, wherein 3D represents the 3D tumor model and 2D represents the 2D tumor model. As can be seen from FIG. 19, most of the anti-cancer drugs had strong inhibitory effect (inhibition rate > 50%) on the 2D tumor model at 10. mu.M, but showed no effect (inhibition rate < 50%) on the 3D model, and the in vivo experiments using doxorubicin hydrochloride, epirubicin, and mitomycin as examples confirmed that the anti-cancer drugs were effective on H460 cell line transplantable tumors in mice. In vivo experiments with gemcitabine as an example demonstrated that the anticancer drug was not effective against H460 cell line transplantable tumors consistent with the 3D tumor model. And evaluating the 3D screening platform according to the anti-cancer drug screening data, wherein the CV obtained by the calculation result is less than 0.1 and the Z factor calculation result is more than 0.5, which indicates that the 3D high-throughput screening platform can be used for high-throughput drug screening.
Wherein, the fluorescence result chart of the tumor cell line 3D high flux drug action live/dead cell identification of 3D-IV-1 (H460) is shown in figure 20. As can be seen from FIG. 20, under the effect of adriamycin, tumor cells of the 3D model die obviously, and the result also shows that the 3D tumor organ microchip can be matched with a high content imaging system for drug screening.
It should be understood that the above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily think of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (10)
1. A3D high-flux organ microchip is characterized by comprising a liquid storage layer, a 3D culture layer and a bottom plate layer which are sequentially arranged in a layered manner;
the liquid storage layer is provided with a plurality of liquid storage through holes, and the liquid storage through holes are used for storing culture liquid;
a 3D culture layer having a plurality of culture microwells for 3D cell culture;
the liquid storage through holes correspond to the culture micropores one by one.
2. The organ microchip according to claim 1, wherein the pore diameter of the reservoir through-hole is greater than or equal to the pore diameter of the culture micro-hole.
3. The organ microchip of claim 1, wherein the reservoir through hole of the reservoir layer is a reservoir well.
4. The organ microchip according to claim 1, wherein a plurality of the liquid storage through holes constitute a through hole liquid storage region, and liquid containing grooves are formed on a liquid storage layer around the through hole liquid storage region.
5. The 3D high-throughput organ microchip according to claim 1, wherein the reservoir through hole of the reservoir layer and/or the culture micro-hole of the 3D culture layer are straight holes.
6. The 3D high-flux organ microchip according to claim 1, wherein the surface of the substrate layer adjacent to the 3D culture layer has hydrophobicity.
7. The 3D high-throughput organ microchip according to claim 6, wherein an uneven rough surface is formed on the surface of the substrate layer corresponding to the culture wells of the 3D culture layer.
8. The 3D high-flux organ microchip according to claim 7, wherein the rugged rough surface is a scratch pattern rough surface.
9. The 3D high-throughput organ microchip according to any one of claims 1 to 8, further comprising a first two-sided glue layer disposed between the reservoir layer and the 3D culture layer and a second two-sided glue layer between the 3D culture layer and the bottom plate layer.
10. The 3D high flux organ microchip according to any one of claims 1 to 8, wherein the organ microchip is integrally formed.
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