CN114350514B - Multicellular chain culture device and application thereof in preparation of hepatic cable structure - Google Patents
Multicellular chain culture device and application thereof in preparation of hepatic cable structure Download PDFInfo
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Abstract
The invention discloses a multicellular chain-shaped culture device and application thereof in preparing a hepatic cable structure, and belongs to the field of cell culture devices. The multicellular chain culture device and the liver rope structure culture method can effectively improve the cell culture dimension and the information interconnection between cells so as to provide the function of in-vitro cell culture.
Description
Technical Field
The invention relates to the technical field of manufacturing of cell culture chips, in particular to a multicellular chain culture device and application thereof in preparing a hepatic cable structure.
Background
The liver is the largest digestive organ of the human body and plays the role of digesting most exogenous substances in the human body. The liver has complex physiological structure and a special double blood supply system, a part of blood absorbs oxygen from lung tissues and flows into the liver through hepatic artery, and a part of blood absorbs nutrient substances through digestive system and flows into the liver through portal vein and flows into the liver through abundant microvasculature. The liver has many liver lobules inside, which are the basic unit of liver composition. The hepatic lobule is hexagonal, a central vein running along its long axis is arranged in the center, and hepatic chordae and hepatic blood sinus are radially arranged around the central vein. The single layer of liver cells is arranged into a rugged plate-shaped structure, which is called a liver plate, and the section of the liver plate is in a rope shape, which is called a hepatic rope. The modern medical engineering is difficult to etch the complex structure of the liver in vitro, most of bionic organs are built in the complex engineering structure, and the complexity of the traditional two-dimensional planar culture is increased by a device capable of being designed and processed or a polymer material with a certain physiological function to simulate the human organs, so that part of known physiological functions are realized. However, the cells or tissues obtained in these ways still differ greatly from the human body, and many functions of the human body are still obtained in a spontaneous form rather than passively.
Conventional planar culture cell attachment is often based on limited intercellular membrane contact and the intercellular coupling is very limited. In recent years, considerable research has been devoted to increasing the dimensions of cell culture to enhance the information exchange of cells to increase the in vitro culture function of cells, such as forming a stereoscopic sphere by cell self-assembly, enhancing cell-to-cell contact; hydrogel is added into the culture system to achieve the effect of improving the cell culture dimension so as to enhance the interaction between cells. However, these approaches have limitations in that spherically cultured cells can be hindered by nutrient and oxygen transport, resulting in cell death in the central region of the sphere, and thus the sphere diameter is limited to below 200 μm, which is far from the real liver. How to effectively increase the three-dimensional culture volume of cells and ensure the material delivery is a great difficulty in the tissue engineering of various large organs.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a multicellular chain culture device and application thereof in preparing hepatic cable structure, which can effectively improve cell culture dimension and information interconnection between cells to provide a function of in vitro culture of cells.
To achieve the above and other related objects, an aspect of the present invention provides a multicellular chain-like culture apparatus, including a culture apparatus body and a microwell array module provided to the culture apparatus body, the microwell array module including at least two rows of microwell columns;
each row of micropore row is provided with a plurality of micropores which are arranged in sequence, and a communication channel for communicating the two adjacent micropores is arranged between the two adjacent micropores.
In some embodiments of the invention, the microwells are cylindrical microwells;
preferably, the diameter of the micropores is 200-2000 microns, and the depth of the micropores is 200-5000 microns.
In some embodiments of the invention, the spacing between two adjacent microwells in each row of microwell columns is 2 to 10 microns.
In some embodiments of the present invention, the hole walls of two adjacent micropores in each row of the micropore columns are provided with notches for forming communication channels;
preferably, the opening degree of the notch is gradually increased from the bottom of the micropore to the direction of the mouth of the micropore;
more preferably, the notch is a V-shaped notch.
In some embodiments of the present invention, each of the micropores in two adjacent rows of the micropore array module are disposed in sequence opposite to each other;
preferably, each micropore in two adjacent rows of micropore columns is arranged oppositely in turn, and the distance between the two oppositely arranged micropores is 10-1000 microns.
In some embodiments of the invention, the microwell array module comprises at least one set of microwell columns; the group of micropore columns at least comprises two rows of micropore columns which are adjacently arranged and are used for cell chain culture;
preferably, the spacing between each of the groups of columns of microwells is 50-50000 microns;
and/or, the micro-hole columns in each group of micro-hole columns are arranged in a linear array or a curve array;
and/or the curve array arrangement is selected from at least one of circular array arrangement, elliptic array arrangement, semicircular array arrangement, triangular array arrangement, trapezoid array arrangement or polygonal array arrangement, or other needed patterns are arranged.
In some embodiments of the invention, each of the microwells is treated with a surface-hydrophobizing agent, preferably agarose or Pluronic F-127;
and/or the surface of the micropore array module is coated with a modified material, wherein the modified material is a material which has good biocompatibility, good optical permeability, easy molding and hydrophobicity; preferably, the modifying material is selected from one or more of agarose, polyethylene glycol or sodium alginate;
and/or the micropore array module adopts a plastic material with thermosetting molding, injection molding or photo-curing molding, preferably, the plastic material is selected from one or a plurality of combinations of polymethyl acryl gelatin (GelMA), polymethoxy siloxane (PDMS), polyethylene glycol (PEG), polymethyl methacrylate (PMMA) and polyethylene terephthalate (PET).
The second aspect of the present invention provides a method for preparing a multicellular chain culture device, comprising the steps of:
s1, manufacturing a micropore array module design diagram by adopting computer graphic design software;
s2, preparing a micropore array module mould or a micropore array module by adopting a photoetching, three-dimensional printing process or a pouring molding process;
s3, pouring a prefabricated plastic material with thermosetting molding, injection molding or photo-curing molding into the micropore array mold, and solidifying and pouring to obtain a micropore array module;
the plastic material is at least one selected from GelMA, PDMS, PEG, PMMA or PET;
s4, performing hydrophobization treatment on micropores of the micropore array module by using a hydrophobization treatment reagent.
A third aspect of the invention is the use of a multicellular chain culture device in at least one of the following features:
a1, application in multicellular culture of tissues or organs;
a2, application in multicellular culture of tissue or organ similar to hepatic chordae structure;
a3, application in multicellular culture of a tissue or an organ with a hepatic cable structure;
a4, application of the liver cable structure in preparation of bionic liver lobule structures.
The fourth aspect of the present invention provides a multi-cell chain culture method for a hepatic cable or hepatic cable-like structure, using the above multi-cell chain culture apparatus, the culture method comprising the steps of:
s100, preparing a suspension of a first cell and a suspension of a second cell respectively; the first cells are selected from one or more of liver parenchymal cells, liver cells formed by differentiation of pluripotent stem cells and primary liver cells which are digested and separated by liver cancer tissues of a patient; the second cells are selected from one or more of liver sinus endothelial cells, endothelial cells isolated by digestion of liver cancer tissue of the patient;
preferably, the first cells with the cell fusion degree reaching 80% -90% and the second cells with the cell fusion degree reaching 80% -90% are respectively prepared into cell suspensions;
preferably, the first cell and the second cell are selected from the group consisting of organs having a chain-like cellular structure; more preferably, the organ provided with a chain cell structure is selected from liver, intestinal tract or lung;
s200, mixing the first cells and the first cells according to a specific proportion, and adjusting the cell concentration of the cell suspension to be 5 multiplied by 10 < 4 > -1 multiplied by 10 < 7 >/mL; wherein, the specific proportion is that two kinds of cells are mixed according to the specific proportion of the cell number in the organ, and the mixing proportion is 3:1 or 4:1 or 10:1 or other proportions of particular significance;
s300, adding 1% -20% of matrigel material into the cell suspension, and uniformly mixing; preferably, the matrigel material is gel with a bracket function; more preferably, the Matrigel material is Matrigel;
s400, adding the cell suspension obtained in the S400 into micropores in a micropore array module of the multicellular chain culture device; preferably, 5000 to 100000 cells are placed in each microwell;
s500, culturing to obtain a hepatic cable or hepatic cable-like structure.
Drawings
FIG. 1 is a schematic diagram of a multicellular chain culture device placed in a chip or well plate for cell chain culture;
FIG. 2 is a partial micro-hole array module scanning electron microscope with V-shaped notch to a scale of 100 microns; wherein A is a partial scanning electron microscope image of a group of micropore columns, and B is a scanning electron microscope image of two adjacent micropores with V-shaped notches;
FIG. 3 is a statistical diagram of cell mass culture and chain culture protein expression;
FIG. 4 is an optical image of a multicellular chain culture device.
Reference numerals in the drawings:
100. a culture vessel; 200. a microwell array module; 201. micropores; 202. v-shaped notch.
Detailed Description
The inventor provides a multicellular chain culture device which is suitable for cell culture of different sources or different tissue types, and the micropore array module can be matched with various commercial culture vessels, fully considers the operation habits of biologists, reduces the learning cost and has high user friendliness. In addition, the micropore array module has adaptability with the existing cell culture pore plate, and has good compatibility with the existing biological analysis and imaging instrument. Cell chain culture further improves the compactness of cell connection on the basis of cell spheroid culture, and information interconnection between cells is enhanced. On this basis, the present invention has been completed.
The invention provides a multicellular chain-shaped culture device, which comprises a culture device body and a micropore array module arranged on the culture device body, wherein the micropore array module comprises at least two rows of micropore columns, each row of micropore columns is provided with a plurality of micropores which are sequentially arranged, and a communication channel for communicating two adjacent micropores is arranged between the two adjacent micropores. The communication channel is preferably formed by opening notches in the hole walls of two adjacent micropores in each row of micropore columns, preferably, the opening degree of the notches gradually increases from the bottom of the micropores to the direction of the mouth of the micropores, and more preferably, the notches are V-shaped notches.
The information interaction of two adjacent cell clusters can be increased through the communication channel, and the formation of a hepatic cable structure is accelerated. The micropores are subjected to surface hydrophobization treatment, and the hydrophobizing reagent is a reagent which has good biocompatibility, good optical permeability and easy operation, such as agarose, pluronic F-127 and other reagents which can change the surface adsorption performance so as to reduce the adhesion of cells to the culture wall surface.
In a preferred embodiment, the micropores are cylindrical micropores having a diameter of 200 to 2000 microns, alternatively 200 to 500 microns, 500 to 700 microns, 700 to 1000 microns, 1000 to 2000 microns; the depth of the micropores is 200-5000 microns, and the proper diameter and depth of the micropores can stably form cell spheres with the diameter range of about 200-300 microns.
In a preferred embodiment, the spacing between two adjacent micro-holes in each row of micro-hole columns is 2-200 micrometers, preferably 5-10 micrometers, optionally 2-10 micrometers, 10-50 micrometers, 50-100 micrometers, 100-200 micrometers, and the specific spacing between two adjacent micro-holes refers to the minimum distance between two micro-holes, more specifically, the wall thickness between two adjacent micro-holes, so that a certain wall thickness is ensured to form a V-shaped notch.
In a preferred embodiment, the spacing between two adjacent rows of microwell arrays in each set of microwell array modules is 10 to 1000 microns. The specific distance between two adjacent rows of microwell columns refers to the minimum distance between two rows of microwell columns, and a certain distance is set to form a connected cell chain, so that microwells in different rows of microwell arrays in a connected group.
In a preferred embodiment, the microwell array module comprises at least one set of microwell columns; the group of micropore columns at least comprises two rows of micropore columns which are adjacently arranged and are used for cell chain culture. Preferably, the spacing between the columns of microwells of each set is 50-5000 microns, optionally 50-100 microns, 100-500 microns, 500-1000 microns, 1000-2000 microns, 2000-3000 microns, 3000-5000 microns. Specifically, the spacing between the groups of microporous columns refers to the minimum distance between the groups, and a wider distance is arranged between each group, so that independent growth can be carried out between different cell chains without mutual interference.
The micro-hole columns in each group of micro-hole columns are arranged in a linear array or a curve array; the curve array arrangement is at least one selected from circular array arrangement, elliptic array arrangement, semicircular array arrangement, triangular array arrangement, trapezoid array arrangement or polygonal array arrangement.
The using method of the multicellular chain culture device comprises the following steps: the first culture vessel 100 is placed in culture, and the culture vessel can be a micro flow channel chip with a liquid circulation function or can be designed to be prepared into different sizes so as to be directly placed into the micro flow channel chip or a cell culture plate 100, wherein the culture plate comprises one of a 96-well plate, a 48-well plate, a 24-well plate, a 12-well plate, a 6-well plate, a 3.5cm culture dish, a 6cm culture dish and a 10cm culture dish; and secondly, cell chain culture can be carried out by directly dripping cell suspension or carrying out fluid delivery through micropores into a micropore array module.
In a preferred embodiment, each of the microwells is treated with a surface-hydrophobizing agent, preferably Pluronic F-127, to reduce cell adhesion to the culture wall, and has good biocompatibility, good optical permeability, and ease of handling. Preferably, the micropore array module is made of at least one material such as GelMA, PDMS, PEG, PMMA, PET.
The application of the multicellular chain culture device in preparing the hepatic chordae structure in the bionic hepatic lobule structure is suitable for cell culture of different sources or different tissue types, the micropore array module can be matched with various commercially available culture vessels, the operation habits of biologists are fully considered, the learning cost is reduced, and the method has high user friendliness. In addition, the micropore array module has adaptability with the existing cell culture pore plate, and has good compatibility with the existing biological analysis and imaging instrument. Cell chain culture further improves the compactness of cell connection on the basis of cell spheroid culture, and information interconnection between cells is enhanced.
The multicellular chain culture device is suitable for co-culturing two or more than two cells in the hepatic cable structure of liver organs, and can be specifically liver parenchymal cells, liver cells formed by differentiation of pluripotent stem cells, primary liver cells digested and separated by liver cancer tissues of patients, liver sinus endothelial cells, endothelial cells digested and separated by liver cancer tissues of patients and other organs or tissue cells. The device is not limited to the application of hepatic cable structure, but is also applicable to other multicellular cultures with similar structural tissues or organs.
The advantageous effects of the present invention are further illustrated below with reference to examples.
In order to make the objects, technical solutions and advantageous technical effects of the present invention more clear, the present invention is described in further detail below with reference to examples. However, it should be understood that the examples of the present invention are merely for the purpose of explaining the present invention and are not intended to limit the present invention, and the examples of the present invention are not limited to the examples given in the specification. The specific experimental or operating conditions were not noted in the examples and were made under conventional conditions or under conditions recommended by the material suppliers.
Furthermore, it is to be understood that the reference to one or more method steps in this disclosure does not exclude the presence of other method steps before or after the combination step or the insertion of other method steps between these explicitly mentioned steps, unless otherwise indicated; it should also be understood that the combined connection between one or more devices/means mentioned in the present invention does not exclude that other devices/means may also be present before and after the combined device/means or that other devices/means may also be interposed between these two explicitly mentioned devices/means, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the method steps is merely a convenient tool for identifying the method steps and is not intended to limit the order of arrangement of the method steps or to limit the scope of the invention in which the invention may be practiced, as such changes or modifications in their relative relationships may be regarded as within the scope of the invention without substantial modification to the technical matter.
In the examples described below, reagents, materials and apparatus used are commercially available unless otherwise specified.
Example 1
As shown in fig. 1 and 2, the microwell array module 200 is placed in the culture vessel 100, and the microwell array module 200 is provided with six rows of microwells 201 arranged in sequence, and each row of microwells 201 is arranged in a circular shape. Two adjacent rows are a group of micro-hole columns, three groups of micro-hole columns are all arranged, the three groups of micro-hole columns form circular arrays with different radiuses at intervals of about 50-50000 microns, each group has a wider distance, and different cell chains can independently grow without mutual interference.
Wherein, the diameter of the micropore 201 is 300 micrometers, the distance between two adjacent micropores 201 in each row of micropore columns with the depth of 250 micrometers is 5 micrometers, and the hole walls of two adjacent micropores 201 in each row of micropore columns are provided with V-shaped notches 202. Each of the microwells 201 in the adjacent two rows of microwell columns in the microwell array module 200 is disposed opposite to each other in sequence, and the interval between the two oppositely disposed microwells 201 is 10 micrometers.
The array module 200 is obtained using polymethoxy siloxanes and by soft lithography or 3D printing.
Each of the microwells was treated with 0.5% Pluronic F-127 deionized water.
Example 2
The preparation method of the multicellular chain culture device comprises the following steps:
(1) Using computer graphic design software to perform layout graphic design on the needed micropore array module to form a micropore array module design drawing, and patterning the photosensitive photoresist through a soft lithography process to form a photoresist film (micropore array module mould) with a micro-column array;
(2) Degassing the prepared PDMS (polymethoxy siloxane) mixture, pouring the mixture into the photoresist mold, and after standing for a while, heating and drying the mixture, and carefully stripping the mixture from the grinding tool after the PDMS is completely solidified to form a PDMS module with a micropore array and V-shaped grooves;
(3) Cutting PDMS according to a designed pattern, and adapting to a microfluidic channel chip or cell culture plates with different sizes to obtain a multicellular chain culture device;
(4) Soaking the multicellular chain culture device for not less than 3 hours by using a hydrophobizing reagent (containing 0.5% Pluronic F-127 deionized water) to carry out surface hydrophobizing treatment of the device;
(5) Excess hydrophobizing reagent was removed from the surface by washing with PBS and the device surface incubation was performed for at least half an hour using complete medium of cell culture for co-cell culture.
Example 3
Multicellular chain culture method for hepatic chordae structure in bionic hepatic lobular structure
(1) Provides a liver cancer cell (HepG 2) and an endothelial cell line (EAhy 926) which stably grow, and the cell fusion degree reaches 80-90 percent.
(2) Endothelial cells and liver cancer cells were mixed according to a specific ratio of 4:1, mixing, performing cell counting, and adjusting the cell concentration of the cell suspension to be 5X 10-4 to 1X 10-7 cells/mL.
(3) And adding 1% -20% Matrigel into the cell suspension, and uniformly blowing by using a pipetting gun.
(4) The cell suspension was added to the device using a pipette gun to drop 5000-100000 cells uniformly in each microwell.
(5) Placing into a cell culture box for stable culture for at least 24 hours, and enabling the cells to self-assemble to form chains, and then changing the liquid every 24 hours.
(6) Albumin ALB and endothelial cell marker CD31 secretion analysis was performed by culturing until the fifth day, immunofluorescence results were obtained by microscopy, and statistical analysis was performed using Image J, and the results are shown in fig. 3 and 4. As can be seen from the results shown in FIG. 3, the chain structure of the cells in the chain culture has higher albumin expression, and the functional effect of the cell culture is obviously better than that of the cell culture in the spherical culture.
The foregoing embodiments are merely illustrative of the principles of the present application and their effectiveness, and are not intended to limit the application. Modifications and variations may be made to the above-described embodiments by those of ordinary skill in the art without departing from the spirit and scope of the present application. Accordingly, it is intended that all equivalent modifications and variations which may be accomplished by persons skilled in the art without departing from the spirit and technical spirit of the disclosure be covered by the claims of this application.
Claims (8)
1. A multicellular chain culture device, which is characterized by comprising a culture device body and a micropore array module (200) arranged on the culture device body, wherein the micropore array module (200) comprises at least two rows of micropore columns;
each row of micropore columns is provided with a plurality of micropores (201) which are sequentially arranged, and a communication channel for communicating the two adjacent micropores is arranged between the two adjacent micropores;
the pore walls of two adjacent micropores (201) in each row of micropore columns are provided with notches for forming a communication channel, the opening degree of the notches gradually increases from the bottom of the micropores (201) to the direction of the mouth of the micropores (201), and the notches are V-shaped notches (202); the spacing between two adjacent micropores in each row of micropore columns is 2-200 microns, and the walls between the two adjacent micropores form V-shaped notches (202);
the microwell array module comprises at least one set of microwell columns; the group of micropore columns at least comprises two rows of micropore columns which are adjacently arranged and are used for cell chain culture; the micropore columns in each group of micropore columns are distributed in a curve array;
the spacing between each group of micropore columns is 50-50000 micrometers, and the spacing between every two adjacent micropore columns in the micropore array module is 10-1000 micrometers.
2. The multicellular chain culture device according to claim 1, wherein the microwells (201) are cylindrical microwells;
preferably, the diameter of the micropores (201) is 200-2000 microns, and the depth of the micropores (201) is 200-5000 microns.
3. The multicellular chain culture device of claim 1 wherein;
the curve array arrangement is at least one selected from circular array arrangement, elliptic array arrangement, semicircular array arrangement, triangular array arrangement, trapezoid array arrangement or polygonal array arrangement.
4. The multicellular chain culture device according to claim 1, wherein each microwell (201) is treated with a surface hydrophobic reagent, preferably agarose or Pluronic F-127;
and/or the surface of the micropore array module is coated with a modified material, wherein the modified material is a material which has good biocompatibility, good optical permeability, easy molding and hydrophobicity; preferably, the modifying material is selected from one or more of agarose, polyethylene glycol or sodium alginate;
and/or the micropore array module adopts a plastic material with thermosetting molding, injection molding or photo-curing molding, preferably, the plastic material is selected from one or a plurality of combinations of polymethyl propenyl gelatin, polymethoxy siloxane, polyethylene glycol, polymethyl methacrylate and polyethylene terephthalate.
5. The method for producing a multicellular chain culture device according to any one of claims 1 to 4, comprising the steps of:
s1, manufacturing a micropore array module design diagram by adopting computer graphic design software;
s2, preparing a micropore array module mould or a micropore array module by adopting a photoetching, three-dimensional printing process or a pouring molding process;
s3, pouring a prefabricated plastic material with thermosetting molding, injection molding or photo-curing molding into the micropore array mold, and solidifying and pouring to obtain a micropore array module;
s4, performing hydrophobization treatment on micropores of the micropore array module by using a hydrophobization treatment reagent.
6. Use of the multicellular chain culture device of claims 1-4 in at least one of the following features:
a1, application in multicellular culture of tissues or organs;
a2, application in multicellular culture of tissue or organ similar to hepatic chordae structure;
a3, application in multicellular culture of a tissue or an organ with a hepatic cable structure;
a4, application of the liver cable structure in preparation of bionic liver lobule structures.
7. A method for multicellular chain culture of a hepatic cable or hepatic cable-like structure, characterized by using the multicellular chain culture apparatus according to claim 1-4, the method comprising the steps of:
s100, preparing a suspension of a first cell and a suspension of a second cell respectively;
preferably, the first cells with the cell fusion degree reaching 80% -90% and the second cells with the cell fusion degree reaching 80% -90% are respectively prepared into cell suspensions;
preferably, the first cell and the second cell are selected from the group consisting of organs having a chain-like cellular structure; more preferably, the organ provided with a chain cell structure is selected from liver, intestinal tract or lung;
s200, mixing the first cells and the first cells according to a specific proportion, and adjusting the cell concentration of the cell suspension to be 5 multiplied by 10 < 4 > -1 multiplied by 10 < 7 >/mL;
s300, adding 1% -20% of matrigel material into the cell suspension, and uniformly mixing; preferably, the matrigel material is gel with a bracket function; more preferably, the Matrigel material is Matrigel;
s400, adding the cell suspension obtained in the S400 into micropores in a micropore array module of the multicellular chain culture device; preferably, 5000 to 100000 cells are placed in each microwell;
s500, culturing to obtain a hepatic cable or hepatic cable-like structure.
8. The method according to claim 7, wherein the first cells are selected from at least one of liver parenchymal cells, liver cells formed by differentiation of pluripotent stem cells, and primary liver cells isolated by digestion of liver cancer tissue of a patient;
the second cell is selected from at least one of liver sinusoidal endothelial cells or endothelial cells isolated by digestion of liver cancer tissue of the patient.
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| CN112375681A (en) * | 2020-11-19 | 2021-02-19 | 中国科学院上海微***与信息技术研究所 | Organ chip and application thereof |
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| CN102928584A (en) * | 2003-09-25 | 2013-02-13 | 富山县政府 | Microwell array chip and method of manufacting same |
| CN103421691A (en) * | 2013-07-12 | 2013-12-04 | 西北工业大学 | Glass chip for cultivating single cell array based on microfluidic patterning technology and preparation method thereof |
| CN112375681A (en) * | 2020-11-19 | 2021-02-19 | 中国科学院上海微***与信息技术研究所 | Organ chip and application thereof |
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