CN114350514A - Multi-cell chain culture device and application thereof in preparation of liver cable structure - Google Patents
Multi-cell chain culture device and application thereof in preparation of liver cable structure Download PDFInfo
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Abstract
The invention discloses a multi-cell chain-shaped culture device and application thereof in preparation of a liver cable structure, and belongs to the field of cell culture devices. The multi-cell chain culture device and the liver cable structure culture method can effectively improve the dimension of cell culture and the information interconnection among cells so as to provide the function of in-vitro cell culture.
Description
Technical Field
The invention relates to the technical field of cell culture chip manufacturing, in particular to a multi-cell chain culture device and application thereof in preparation of a liver cable structure.
Background
The liver is the largest digestive organ of the human body and plays the role of digestion of most foreign substances in the human body. The physiological structure of the liver is complex, and a special dual blood supply system is provided, wherein a part of blood absorbs oxygen from lung tissues and flows into the liver through a hepatic artery, and a part of blood absorbs nutrient substances through a digestive system and then is injected into the liver through a portal vein, and flows into the liver through rich capillaries. The liver has many lobules inside, which are the basic units of the liver. The hepatic lobules are hexagonal, the center of the hepatic lobules is provided with a central vein running along the long axis of the hepatic lobules, and hepatic cords and hepatic blood sinuses are radially arranged around the central vein. The liver cells are arranged in a single layer to form an uneven plate-shaped structure, called liver plate, and the section of the liver plate is in a cord shape, called liver cord. Modern medical engineering is difficult to carve out the complex structure of liver again in vitro, and most bionic organs are built in the complex engineering structure, but the device that can design processing or the macromolecular material that possesses certain physiology function increase the complexity of traditional two-dimensional plane culture and simulate human organ, realize some known physiology functions. However, the cells or tissues obtained by these methods are still quite different from those of the human body, and many functions of the human body are still obtained in a spontaneous form rather than passively.
Traditional planar culture cell junctions are often based only on limited intercellular membrane contact and the intercellular junctions are very limited. In recent years, considerable research has been devoted to increasing the dimension of cell culture to enhance the information exchange of cells to increase the in vitro culture function of cells, for example, by forming a three-dimensional sphere through cell self-assembly to enhance the contact between cells; hydrogel is added into the culture system to achieve the effect of improving the dimension of cell culture to enhance the interaction between cells. However, these methods still have limitations, and the cell cultured in the form of spheres will be inhibited from transferring nutrients and oxygen, resulting in cell death in the central area of the spheres, and the diameter of the spheres is limited to 200 μm or less, which is far from the size of real liver. How to effectively increase the three-dimensional culture volume of cells and ensure the material transportation is a great problem to be solved urgently in the tissue engineering of each large organ.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a multi-cell chain culture device and its application in preparing liver cable structure, which can effectively improve the dimension of cell culture and the information interconnection between cells to provide the function of in vitro cell culture.
In order to achieve the above and other related objects, an aspect of the present invention provides a multi-cell chain culture apparatus, including a culture apparatus body and a microwell array module disposed on the culture apparatus body, the microwell array module including at least two rows of microwell rows;
each row of micropore rows are provided with a plurality of micropores which are arranged in sequence, and a communication channel for communicating two adjacent micropores is arranged between two adjacent micropores.
In some embodiments of the invention, the microwells are cylindrical microwells;
preferably, the diameter of the micropores is 200-2000 micrometers, and the depth of the micropores is 200-5000 micrometers.
In some embodiments of the present invention, the distance between two adjacent micropores in each row of micropore columns is 2 to 10 micrometers.
In some embodiments of the present invention, the pore walls of two adjacent micropores in each row of micropore rows are provided with notches for forming communicating channels;
preferably, the opening degree of the notch is gradually increased from the bottom of the micropore to the opening of the micropore;
more preferably, the notch is a V-shaped notch.
In some embodiments of the present invention, each of the microwells in two adjacent rows of the microwell array module are sequentially arranged oppositely;
preferably, each micropore in two adjacent rows of micropore rows is arranged oppositely in sequence, and the distance between two oppositely arranged micropores is 10-1000 microns.
In some embodiments of the invention, the microwell array module comprises at least one set of columns of microwells; the group of micropore rows at least comprises two rows of micropore rows which are arranged adjacently and used for cell chain culture;
preferably, the distance between each group of micropore rows is 50-50000 micrometers;
and/or the micropore columns in each group of micropore 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, elliptical array arrangement, semicircular array arrangement, triangular array arrangement, trapezoidal array arrangement or polygonal array arrangement, or other required graphs.
In some embodiments of the invention, each of the microwells is treated with a surface hydrophobizing agent, preferably, the hydrophobizing agent is agarose or Pluronic F-127;
and/or, the surface of the micropore array module is coated with a modified material, and the modified material is a material with good biocompatibility, good optical permeability, easy molding and hydrophobicity; preferably, the modified 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 photocuring molding, preferably, the plastic material is selected from one or more of polymethylacryl gelatin (GelMA), Polymethoxysiloxane (PDMS), polyethylene glycol (PEG), polymethyl methacrylate (PMMA) and polyethylene terephthalate (PET).
The second aspect of the present invention provides a method for producing a multi-cell chain culture apparatus, comprising the steps of:
s1, manufacturing a micropore array module design drawing by using computer graphic design software;
s2, preparing a micropore array module mould or a micropore array module by adopting photoetching, three-dimensional printing technology or pouring molding technology;
s3, pouring a prefabricated plastic material with thermosetting molding, injection molding or photocuring molding into the micropore array mold, and curing and reversing the mold to obtain a micropore array module;
the plastic material is selected from at least one of GelMA, PDMS, PEG, PMMA or PET;
s4, hydrophobization of the microwells of the microwell array module is performed by using a hydrophobization reagent.
The third aspect of the present invention is an application of the multi-cell chain culture apparatus in at least one of the following features:
a1, use in multicellular culture of a tissue or organ;
a2, use in the multicellular culture of a tissue or organ resembling a hepatic cord structure;
a3, use in the multicellular culture of a tissue or organ of the hepatic duct structure;
a4, and application in preparing a liver cable structure in a bionic liver lobule structure.
In a fourth aspect, the present invention provides a method for multi-cell chain culture of a hepatic duct or hepatic duct-like structure, using the above multi-cell chain culture apparatus, the method comprising the steps of:
s100, respectively preparing a suspension of a first cell and a suspension of a second cell; the first cell is selected from one or more of liver parenchymal cell, liver cell formed by differentiation of pluripotent stem cell, and primary liver cell digested and separated from liver cancer tissue of a patient; the second cell is selected from one or more of liver sinusoidal endothelial cells, endothelial cells digested and separated from liver cancer tissues of the patient;
preferably, a first cell with the cell fusion degree of 80-90% and a second cell with the cell fusion degree of 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 cell structure; more preferably, the organ having a chain-like cell structure is selected from the group consisting of liver, intestinal tract or lung;
s200, mixing the first cell and the first cell according to a specific proportion, and adjusting the cell concentration of the cell suspension to be 5 x 10^ 4-1 x 10^7 cells/mL; wherein, the specific proportion is that according to the specific proportion of the cell number in the organ, the mixing ratio of the two cells is 3: 1 or 4: 1 or 10: 1 or other ratios of particular significance;
s300, adding 1-20% of matrix adhesive material into the cell suspension, and uniformly mixing; preferably, the matrix adhesive material is a gel having a scaffold 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 multi-cell chain culture device; preferably, 5000-100000 cells are placed in each micropore;
s500, obtaining the hepatic cord or hepatic cord-like structure through culture.
Drawings
FIG. 1 is a schematic diagram of a multi-cell chain culture device placed in a chip or a pore plate for cell chain culture;
FIG. 2 is a scanning electron microscope image of a portion of a micropore array module having V-shaped notches, at a scale of 100 microns; wherein A is a scanning electron microscope image of a part of a group of micropore rows, and B is a scanning electron microscope image of two adjacent micropores provided with V-shaped notches;
FIG. 3 is a statistical graph of protein expression in pellet and chain cultures;
FIG. 4 is an optical photograph of a multi-cell chain culture apparatus.
Reference numbers in the figures:
100. a culture vessel; 200. a microwell array module; 201. micropores; 202. v-shaped notches.
Detailed Description
The inventor of the invention provides a multi-cell chain culture device which is suitable for cell culture of different sources or different tissue types, and a micropore array module can be matched with various commercially available culture vessels, fully considers the operation habit 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 instruments. The cell chain culture further improves the connection tightness of cells on the basis of cell spherical culture and strengthens the information interconnection between the cells. On the basis of this, the present invention has been completed.
The invention provides a multi-cell chain-like 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 rows, each row of micropore rows is provided with a plurality of micropores which are sequentially arranged, and a communication channel for communicating two adjacent micropores is arranged between two adjacent micropores. The communicating channel is preferably formed by arranging notches on the hole walls of two adjacent micropores in each row of micropore rows, preferably, the opening degree of each notch is gradually increased from the bottom of each micropore to the opening part of each micropore, and more preferably, each notch is a V-shaped notch.
The information interaction between two adjacent cell groups can be increased through the communication channel, and the formation of the hepatic cord structure is accelerated. The micro-pores are subjected to surface hydrophobization treatment, and the hydrophobization reagent is a reagent with good biocompatibility, good optical permeability and easy operation, such as agarose, Pluronic F-127 and other reagents capable of changing the surface adsorption property so as to reduce the attachment of cells to the culture wall surface.
In a preferred embodiment, the micropores are cylindrical micropores, and the diameter of the micropores is 200 to 2000 micrometers, optionally 200 to 500 micrometers, 500 to 700 micrometers, 700 to 1000 micrometers, 1000 to 2000 micrometers; 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 200-300 microns.
In a preferred embodiment, the distance between two adjacent micropores in each row of micropore rows is 2 to 200 micrometers, preferably 5 to 10 micrometers, optionally 2 to 10 micrometers, 10 to 50 micrometers, 50 to 100 micrometers, and 100 to 200 micrometers, and the specific distance between two adjacent micropores refers to the minimum distance between two micropores, and more specifically, may be the wall thickness between two adjacent micropores, and ensures a certain wall thickness for forming the V-shaped notch.
In a preferred embodiment, the distance between two adjacent rows of micro-hole rows in each group of the micro-hole array module is 10 to 1000 microns. The specific distance between two adjacent rows of micropore rows refers to the minimum distance between two rows of micropore rows, a certain distance is set to form a connecting cell chain, and micropores in different rows of micropore arrays in a connecting group are connected.
In a preferred embodiment, the microwell array module comprises at least one set of columns of microwells; the group of micropore rows at least comprises two rows of micropore rows which are arranged adjacently and used for cell chain culture. Preferably, the distance between each group of micropore rows is 50-5000 micrometers, and can be selected from 50-100 micrometers, 100-500 micrometers, 500-1000 micrometers, 1000-2000 micrometers, 2000-3000 micrometers and 3000-5000 micrometers. Specifically, the distance between the groups of micropore rows refers to the minimum distance between the groups, a wider distance is arranged between each group, and different cell chains can independently grow without mutual interference.
The micropore columns in each group of micropore columns are arranged in a linear array or a curve array; the curved array arrangement is selected from at least one of circular array arrangement, elliptical array arrangement, semicircular array arrangement, triangular array arrangement, trapezoidal array arrangement or polygonal array arrangement.
Method of using the multicellular chain culture apparatus: firstly, placing the mixture in a culture vessel 100 for culture, wherein the culture vessel can be a micro-channel chip with a liquid circulation function or can be designed to prepare different sizes to be directly placed in the micro-channel chip or a cell culture plate 100, and the culture plate comprises one of a 96-hole plate, a 48-hole plate, a 24-hole plate, a 12-hole plate, a 6-hole plate, a 3.5-cm culture plate, a 6-cm culture plate and a 10-cm culture plate; secondly, cell chain culture can be carried out by directly dripping cell suspension or carrying out fluid conveying to the micropore array module through micropores.
In a preferred embodiment, each micropore is treated with a surface hydrophobizing agent, preferably Pluronic F-127 is selected as the hydrophobizing agent to reduce cell adhesion to the culture wall, and the surface hydrophobizing agent has good biocompatibility, good optical permeability and easy operation. Preferably, the micropore array module is made of at least one material of GelMA, PDMS, PEG, PMMA, PET, etc.
The application of the multi-cell chain culture device in the liver cable structure in the preparation of the bionic liver lobule structure is suitable for the cell culture of different sources or different tissue types, the micropore array module can be matched with various commercially available culture vessels, the operation habit of a biologist is 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 instruments. The cell chain culture further improves the connection tightness of cells on the basis of cell spherical culture and strengthens the information interconnection between the cells.
The multi-cell chain culture device is suitable for co-culture of two or more than two cells in liver funicle structures of liver organs, and specifically can be liver parenchymal cells, liver cells formed by differentiation of pluripotent stem cells, primary liver cells digested and separated from liver cancer tissues of patients, liver sinus endothelial cells, endothelial cells digested and separated from liver cancer tissues of patients and other organ or tissue cells. The device is not limited to the application of the hepatic cord structure, but is also suitable for other multi-cell culture with tissues or organs with similar structures.
The following examples are provided to further illustrate the advantageous effects of the present invention.
In order to make the objects, technical solutions and advantageous technical effects of the present invention more clear, the present invention is further described in detail below with reference to examples. However, it should be understood that the embodiments of the present invention are only for explaining the present invention and are not for limiting the present invention, and the embodiments of the present invention are not limited to the embodiments given in the specification. The examples were prepared under conventional conditions or conditions recommended by the material suppliers without specifying specific experimental conditions or operating conditions.
Furthermore, it is to be understood that one or more method steps mentioned in the present invention does not exclude that other method steps may also be present before or after the combined steps or that other method steps may also be inserted between these explicitly mentioned steps, unless otherwise indicated; it is also to be understood that a combined connection between one or more devices/apparatus as referred to in the present application does not exclude that further devices/apparatus may be present before or after the combined device/apparatus or that further devices/apparatus may be interposed between two devices/apparatus explicitly referred to, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.
In the following examples, reagents, materials and instruments used are commercially available unless otherwise specified.
Example 1
A multi-cell chain culture device, as shown in FIG. 1 and FIG. 2, a micropore array module 200 is arranged in a culture vessel 100, the micropore array module 200 is provided with six rows of multiple micropores 201 arranged in sequence, and each row of micropores 201 is arranged in a circle. Two adjacent rows are a group of micropore rows, three groups of micropore rows are provided in total, the three groups of micropore rows form circular arrays with different radiuses at intervals of 50-50000 micrometers, each group has a wider distance, and different cell chains can independently grow without mutual interference.
The diameter of each micropore 201 is 300 micrometers, the depth of each micropore is 250 micrometers, the distance between every two adjacent micropores 201 in each row of micropore rows is 5 micrometers, and the hole walls of every two adjacent micropores 201 in each row of micropore rows are provided with V-shaped notches 202. In the micro-pore array module 200, each micro-pore 201 in two adjacent rows of micro-pore columns is sequentially arranged oppositely, and the distance between two oppositely arranged micro-pores 201 is 10 micrometers.
The array module 200 is obtained by soft lithography or 3D printing using polymethoxysiloxane.
Each of the microwells was treated with 0.5% Pluronic F-127 deionized water.
Example 2
A method for preparing a multi-cell chain culture device, comprising the steps of:
(1) utilizing computer graphic design software to design the layout pattern of the required micropore array module to form a micropore array module design drawing, and patterning the photoresist with photosensitivity through a soft lithography process to form a photoresist film with a micro column array (micropore array module mold);
(2) degassing a prefabricated PDMS (polydimethylsiloxane) mixture, pouring the degassed mixture onto the photoresist mold, performing no vacuum pumping treatment, standing for a while, heating and drying the mixture, and carefully peeling the PDMS from the mold after the PDMS is completely solidified to form a PDMS module with a micropore array and a V-shaped groove;
(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 multi-cell chain culture device;
(4) using a hydrophobization treatment reagent (containing 0.5 percent of Pluronic F-127 deionized water) to soak the multicellular chain culture device for not less than 3 hours to perform device surface hydrophobization treatment;
(5) the surface excess hydrophobizing agent was removed by washing with PBS and the device surface incubation treatment was performed for at least half an hour using complete medium for cell culture, for cocultivation.
Example 3
Multi-cell chain culture method for hepatic chordae structure in bionic hepatic lobule structure
(1) Provides the liver cancer cell (HepG2) and the endothelial cell line (EAhy926) which grow stably, and the cell fusion degree reaches 80 to 90 percent.
(2) Endothelial cells and liver cancer cells are mixed according to a specific ratio of 4: 1, performing cell counting, and adjusting the cell concentration of the cell suspension to 5 x 10^ 4-1 x 10^7 cells/mL.
(3) Adding 1-20% Matrigel into the cell suspension, and uniformly blowing by using a pipette.
(4) The cell suspension was added to the device using a pipette gun so that 5000-100000 cells fell evenly into each microwell.
(5) And (3) stably culturing the cells in a cell culture box for at least 24 hours to enable the cells to form a chain by self-assembly, and replacing the solution every 24 hours.
(6) Albumin ALB and endothelial cell marker protein CD31 secretion analysis was performed by the fifth day of culture, immunofluorescence results were obtained by microscopy and statistical analysis was performed using Image J, the results are shown in fig. 3 and 4. As shown in FIG. 3, it can be seen that the cell chain structure of the chain culture has higher albumin expression, and the cell culture function effect is obviously better than that of the spherical culture.
The above embodiments are merely illustrative of the principles and utilities of the present application and are not intended to limit the application. Any person skilled in the art can modify or change the above-described embodiments without departing from the spirit and scope of the present application. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical concepts disclosed in the present application shall be covered by the claims of the present application.
Claims (10)
1. A multi-cell chain culture device 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 rows;
each row of micropore rows are provided with a plurality of micropores (201) which are arranged in sequence, and a communication channel for communicating two adjacent micropores is arranged between two adjacent micropores.
2. The multicellular chain culture apparatus of 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 apparatus as set forth in claim 1, wherein the distance between two adjacent micropores (201) in each row of the micropore rows is 2 to 200 microns;
and/or the distance between two adjacent rows of micropore rows in the micropore array module is 10-1000 microns.
4. The multicellular chain culture apparatus as claimed in claim 3, wherein the hole walls of two adjacent micropores (201) in each row of micropore rows are provided with notches for forming communicating channels;
preferably, the opening degree of the notch is gradually increased from the bottom of the micropore (201) to the opening direction of the micropore (201);
more preferably, the notch is a V-shaped notch (202).
5. The multi-cell chain culture apparatus according to claim 1 or 4, wherein the microwell array module comprises at least one set of microwell columns; the group of micropore rows at least comprises two rows of micropore rows which are arranged adjacently and used for cell chain culture;
preferably, the distance between each group of micropore rows is 50-50000 micrometers;
and/or the micropore columns in each group of micropore 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, elliptical array arrangement, semicircular array arrangement, triangular array arrangement, trapezoidal array arrangement or polygonal array arrangement.
6. The multi-cell chain culture apparatus according to claim 1, wherein each of the micropores (201) 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, and the modified material is a material with good biocompatibility, good optical permeability, easy molding and hydrophobicity; preferably, the modified 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 photocuring molding, preferably, the plastic material is selected from one or more of polymethylpropenyl gelatin, polymethoxysiloxane and polyethylene glycol, polymethyl methacrylate and polyethylene terephthalate.
7. The method for producing a multi-cell chain culture apparatus according to any one of claims 1 to 6, comprising the steps of:
s1, manufacturing a micropore array module design drawing by using computer graphic design software;
s2, preparing a micropore array module mould or a micropore array module by adopting photoetching, three-dimensional printing technology or pouring molding technology;
s3, pouring a prefabricated plastic material with thermosetting molding, injection molding or photocuring molding into the micropore array mold, and curing and reversing the mold to obtain a micropore array module;
s4, hydrophobization of the microwells of the microwell array module is performed by using a hydrophobization reagent.
8. Use of a multi-cell chain culture device according to claims 1-6 for at least one of the following technical features:
a1, use in multicellular culture of a tissue or organ;
a2, use in the multicellular culture of a tissue or organ resembling a hepatic cord structure;
a3, use in the multicellular culture of a tissue or organ of the hepatic duct structure;
a4, and application in preparing a liver cable structure in a bionic liver lobule structure.
9. A multi-cell chain culture method of hepatic cords or hepatic cord-like structures, using the multi-cell chain culture apparatus according to claims 1 to 6, the culture method comprising the steps of:
s100, respectively preparing a suspension of a first cell and a suspension of a second cell;
preferably, a first cell with the cell fusion degree of 80-90% and a second cell with the cell fusion degree of 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 cell structure; more preferably, the organ having a chain-like cell structure is selected from the group consisting of liver, intestinal tract or lung;
s200, mixing the first cell and the first cell according to a specific proportion, and adjusting the cell concentration of the cell suspension to be 5 x 10^ 4-1 x 10^7 cells/mL;
s300, adding 1-20% of matrix adhesive material into the cell suspension, and uniformly mixing; preferably, the matrix adhesive material is a gel having a scaffold 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 multi-cell chain culture device; preferably, 5000-100000 cells are placed in each micropore;
s500, obtaining the hepatic cord or hepatic cord-like structure through culture.
10. The method for multi-cell chain culture of a liver cable or a liver cable-like structure according to claim 9, wherein the first cell is at least one selected from the group consisting of parenchymal liver cells, hepatocytes formed by differentiation of pluripotent stem cells, and primary hepatocytes isolated by digestion of liver cancer tissue of a patient;
the second cell is selected from at least one of antral endothelial cells or endothelial cells digested and isolated from liver cancer tissue of the patient.
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