CN114540305A - Preparation method of organoid structure based on microfluidic technology high-flux culture - Google Patents
Preparation method of organoid structure based on microfluidic technology high-flux culture Download PDFInfo
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Abstract
The invention belongs to the technical field of organoid preparation, in particular to a preparation method of organoid structure based on microfluidic high-throughput culture, which comprises a cell culture substrate, a microfluidic chip, a microfluidic control system, a high-throughput micro-drug library modeling module and a microfluidic high-throughput drug screening module, and specifically comprises the following steps: step 1: obtaining organoid tissue samples and cutting them into tissue pieces suitable for mechanical grinding; step 2: grinding the tissue block by a mechanical grinding method, and performing first-stage filtration by a first-stage filter screen with the aperture of 80-200 μm to form first-stage filtrate; and step 3: and (3) performing second-stage filtration on the first-stage filtrate by using a second-stage filter screen with the pore size of 30-60 mu m. The proliferation capacity of the organoid treated by the method is obviously stronger than that of the organoid treated by the enzymolysis method, and the mechanical method can obviously enrich tumor cells, remove normal cells and obtain the tumor organoid with higher purity.
Description
Technical Field
The invention relates to the technical field of organoid preparation, in particular to a preparation method of an organoid structure based on microfluidic high-flux culture.
Background
Organoids are a type of organ that uses adult stem cells to reproduce part of the physiological functions of an organ, with 3D cell cultures cultured in vitro having highly similar histological features to the corresponding human organ. With the development of related research, organoids have become the focus of research in the scientific community.
The organoid research is in accordance with the current requirements of individual accurate medical design, has outstanding prospects particularly in the fields of organoid generation mechanisms, related drug screening and model establishment, assists in accelerating the development of new drugs, and is a powerful tool for new drug development. The application of the organoids can obtain expected curative effect and medication result through intelligent analysis and search related cause guidance as a cause exploration path.
The microfluidic technology is a technology for controlling fluid in a micron-scale space, is paid attention to researchers by virtue of the advantages of high flux, high sensitivity, low consumption and the like, and is an emerging interdiscipline related to chemistry, fluid physics, microelectronics, new materials, biology and biomedical engineering. Microfluidic technology mainly applies laminar and droplet phenomena of fluids. Microfluidics enables the preparation of highly monodisperse droplet emulsions at very high throughput. Common microchannel structures are T-type and psi-type. In some cases, aqueous liquids containing different high molecular polymers may also form immiscible droplets in the microfluidic channel.
Compared with tumor cell lines and mouse transplantation models of human tumor tissues, the tumor organoid can be infinitely proliferated under in-vitro culture conditions, so that tumor heterogeneity is well maintained, and meanwhile, gene editing operations including gene knockdown, over-expression and mutation can be performed on the tumor organoid, so that successful establishment of the organoid has important significance for tumor research.
Currently, tumor organoids are obtained mainly by enzymatic digestion of tissues using digestive enzymes to obtain organoid-forming cell aggregates. However, the digestion of tissues by the enzymatic hydrolysis method destroys intercellular junctions, and the digestion time is long, so that the cell activity is reduced, the success rate of sample treatment is low, the number of obtained organoids is small, and the requirements of clinical application and scientific research are difficult to meet.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to solve the following problems: the enzymolysis method is used for digesting tissues to destroy intercellular connection, has longer digestion time, reduces cell activity and causes the technical problem of low success rate of sample processing.
In order to solve the technical problems, the invention adopts the following technical scheme: a preparation method of an organoid structure based on microfluidic technology high-throughput culture, 1, comprises a cell culture substrate, a microfluidic chip, a microfluidic control system, a high-throughput micro-drug library modeling module and a microfluidic high-throughput drug screening module, and specifically comprises the following steps:
step 1: obtaining an organoid tissue sample and cutting it into tissue pieces suitable for mechanical grinding;
step 2: grinding the tissue block by a mechanical grinding method, and performing first-stage filtration by a first-stage filter screen with the aperture of 80-200 μm to form first-stage filtrate; the mechanical method of the invention can obviously enrich tumor cells, remove normal cells and obtain tumor organoids with higher purity;
and step 3: performing second-stage filtration on the first-stage filtrate in a second-stage filter screen with the aperture of 30-60 mu m to obtain solids on the second-stage filter screen, and thus obtaining the organoid;
and 4, step 4: respectively introducing the matrix hydrogel containing organoid cells and the fluorine oil into a three-way device to obtain organoid spheres;
the flow rate of the cell-containing matrix hydrogel is 10-40 mu L/min; the flow rate of the fluorine oil is 40-55 mu L/min;
the cell culture substrate is adhered below the microfluidic chip and used for placing the microfluidic chip;
the microfluidic chip is adhered below the microfluidic control system and on the cell culture substrate, and a microwell array in the microfluidic chip is communicated with the microfluidic control system and used for exchanging substances in a pipeline of the microfluidic control system;
the microfluidic control system is adhered on the microfluidic chip, a microfluidic pipeline in the microfluidic control system is communicated with the microfluidic chip, and the microfluidic control system controls microfluid while exchanging substances;
after organoids are generated by micro-fluidic method, the organoids can be simply, rapidly and accurately printed on a culture carrier by combining with a 3D printing platform, the process is not changed along with the change of operators, the size and the structure of the organoids are proper, uniform and controllable, and compared with the condition that the micro-environment in vivo is lack of culture of a 2D cell line, the characteristics of part of tumor are lost, so that the prediction result is inaccurate, and the high-throughput culture method disclosed by the invention has more accurate result.
Preferably, the organoid is wrapped in matrigel and cultured in organoid culture medium, the organoid culture medium comprises a hydrogel layer and an aqueous two-phase system, and the preparation of the aqueous two-phase system comprises the following steps: selecting a double aqueous phase system as a combination of PEG-dextran; the molecular weight range of PEG is as follows: 1000-: 2.5 to 50 percent; dextran molecular weight range: 70k-500kDa, concentration range: 2.5 to 20 percent;
the hydrogel structure body can simulate the structure and the function of extracellular matrix in a physiological environment, and provides a stable and bionic three-dimensional microenvironment.
Preferably, the high-flux micro-drug library modeling module is used for forming drugs which are arranged in a long micro-droplet order and are separated by oil in micro-tubes to obtain a micro-drug library, the microfluidic high-flux drug screening module is used for screening the drugs of the micro-drug library which are sequentially output by the micro-tubes, the cell culture substrate is bonded below the microfluidic chip in a physical bonding manner, and the cell culture substrate and the microfluidic control system or the microfluidic chip are not subjected to material exchange.
Preferably, the microfluidic chip is bonded under the microfluidic control system in a thermal-packaged chemical bond linkage manner, and is bonded on the cell culture substrate in a physical bonding manner; the diameter of the micro-well array in the micro-fluidic chip is 150-800 μm, and the height is 100-300 μm.
Preferably, the hydrogel layer is composed of a natural hydrogel material and/or a synthetic hydrogel material, the synthetic hydrogel material comprises one or more of polypropylene, polystyrene, polyacrylamide, polylactide, polyglycolide, polylactic acid, polylactic-co-glycolic acid, polyhydroxy acid, polylactic-co-glycolic acid, polydimethylsiloxane, polyanhydride, polyacid ester, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate or polyethylene oxide, and the like, the natural hydrogel material comprises gelatin or derivatives thereof, alginate or derivatives thereof, cellulose or derivatives thereof, agar, matrigel, collagen or derivatives thereof, amino acid or derivatives thereof, glycoprotein and derivatives thereof, collagen or derivatives thereof, and the like, One or more of hyaluronic acid or a derivative thereof, chitosan or a derivative thereof, layer connecting protein, fibronectin, fibrin or a derivative thereof, silk fibroin or a derivative thereof, vitronectin, osteopontin, peptide fragment hydrogel and DNA hydrogel;
the proper size of the microstructure body and the porosity of the hydrogel material ensure good material exchange between cells and a culture environment, and are favorable for the long-term stable survival of the cells in the microstructure body. The biological material provides attachment points for cells, and the cells can be adhered and migrated in the microstructure, so that the spatial arrangement and assembly of the cells are facilitated, and the organoid can be conveniently and better proliferated.
Compared with the prior art, the invention has at least the following advantages:
1. compared with the treatment mode of enzymolysis, the first passage time of the organoids is generally 1-3 weeks, the number of the obtained tumor organoids is small, the time for treating tissues by the enzymolysis method is long, and the activity of the organoids can be damaged, but the number of the organoids obtained by the method is large, the activity of the organoids cannot be damaged, and the drug sensitivity test can be carried out only by culturing for 2-3 days; the proliferation capacity of the organoid treated by the method is obviously stronger than that of the organoid treated by the enzymolysis method, and the mechanical method can obviously enrich tumor cells, remove normal cells and obtain the tumor organoid with higher purity, and a hydrogel structure body provided in a culture medium when the organoid is cultured can simulate the structure and the function of extracellular matrix in a physiological environment and provide a stable and bionic three-dimensional microenvironment. The proper size of the microstructure body and the porosity of the hydrogel material ensure good material exchange between cells and a culture environment, and are favorable for the long-term stable survival of the cells in the microstructure body. The biological material provides attachment points for cells, and the cells can be adhered and migrated in the microstructure, so that the spatial arrangement and assembly of the cells are facilitated, and the organoid can be conveniently and better proliferated.
2. The organoid is used as biological ink, the phenomenon of liquid drops is utilized, the organoid is generated through microfluidics and then combined with a 3D printing platform, the organoid can be simply, conveniently, quickly and accurately printed into a culture carrier, the process does not change along with the change of operators, the size and the structure of the organoid are proper, uniform and controllable, and compared with the condition that the microenvironment in vivo lacks in 2D cell line culture, partial characteristics of the tumor are lost, so that the prediction result is inaccurate, and the high-throughput culture method disclosed by the invention has a more accurate result.
3. The device adopting the cell culture substrate, the microfluidic chip and the microfluidic control system has simple structure, is convenient and controllable to operate, overcomes the defect that the size of the organoid is difficult to be uniform due to uncontrollable performance in the operation process, has uniform micropore arrays with consistent quantity in each microfluidic chip, and solves the problem of inconsistent number of the organoids in each hole.
Drawings
FIG. 1 is a structural diagram of a method for preparing an organoid structure based on microfluidic high-throughput culture according to the present invention;
FIG. 2 is a diagram of the overall process flow of the method for preparing organoid structure based on microfluidic high-throughput culture according to the present invention;
FIG. 3 is a structural diagram of a microfluidic control method for preparing an organoid structure based on microfluidic high-throughput culture according to the present invention;
FIG. 4 is a schematic structural diagram of an organoid medium of the present invention for preparing a organoid structure based on microfluidic high throughput culture;
FIG. 5 is a schematic structural diagram of a hydrogel layer of the method for preparing a organoid structure based on microfluidic high-throughput culture according to the present invention.
Detailed Description
The technical solution of the present patent will be described in further detail with reference to the following embodiments.
Reference will now be made in detail to embodiments of the present patent, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative only for the purpose of explaining the present patent and are not to be construed as limiting the present patent.
In the description of this patent, it is to be understood that the terms "center," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like are used in the orientations and positional relationships indicated in the drawings for the convenience of describing the patent and for the simplicity of description, and are not intended to indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and are not to be considered limiting of the patent.
In the description of this patent, it is noted that unless otherwise specifically stated or limited, the terms "mounted," "connected," and "disposed" are to be construed broadly and can include, for example, fixedly connected, disposed, detachably connected, disposed, or integrally connected and disposed. The specific meaning of the above terms in this patent may be understood by those of ordinary skill in the art as appropriate.
Referring to fig. 1-2, a method for preparing an organoid structure based on microfluidic high-throughput culture, comprising a cell culture substrate, a microfluidic chip, a microfluidic control system, a high-throughput micro-drug library modeling module and a microfluidic high-throughput drug screening module, specifically comprising the following steps:
step 1: obtaining organoid tissue samples and cutting them into tissue pieces suitable for mechanical grinding;
step 2: grinding the tissue blocks by a mechanical grinding method, and performing first-stage filtration by a first-stage filter screen with the aperture of 80-200 μm to form first-stage filtrate;
and 3, step 3: performing second-stage filtration on the first-stage filtrate in a second-stage filter screen with the aperture of 30-60 μm to obtain solid on the second-stage filter screen, and thus obtaining organoids;
and 4, step 4: respectively introducing the matrix hydrogel containing organoid cells and the fluorine oil into a three-way device to obtain organoid spheres;
the flow rate of the cell-containing matrix hydrogel is 10-40 mu L/min; the flow rate of the fluorine oil is 40-55 mu L/min;
the cell culture substrate is adhered below the microfluidic chip and used for placing the microfluidic chip;
the microfluidic chip is adhered below the microfluidic control system and on the cell culture substrate, and a microwell array in the microfluidic chip is communicated with the microfluidic control system and used for exchanging substances in a pipeline of the microfluidic control system;
the microfluid control system is bonded on the microfluid chip, a microfluid pipeline in the microfluid control system is communicated with the microfluid chip, and the microfluid is controlled while the material exchange is carried out; the output end of the microfluid control system is connected with a 3D printing platform;
after organoids are generated by micro-fluidic method, the organoids can be simply, rapidly and accurately printed on a culture carrier by combining with a 3D printing platform, the process is not changed along with the change of operators, the size and the structure of the organoids are proper, uniform and controllable, and compared with the condition that the micro-environment in vivo is lack of culture of a 2D cell line, the characteristics of part of tumor are lost, so that the prediction result is inaccurate, and the high-throughput culture method disclosed by the invention has more accurate result.
Referring to fig. 3, the control of microfluidics while performing the material exchange specifically includes: the perfusion of cells or tissues, the perfusion and replacement of culture media, the inflow and discharge of drugs during drug testing, and the control of microfluid required by experiments are performed.
Referring to fig. 4, organoids are wrapped in matrigel and cultured in organoid culture medium, which includes hydrogel layer and aqueous two-phase system, and the aqueous two-phase system is prepared: selecting a double aqueous phase system as a combination of PEG-dextran; PEG molecular weight range: 1000-: 2.5 to 50 percent; dextran molecular weight range: 70k-500kDa, concentration range: 2.5 to 20 percent.
Referring to fig. 1, the high-throughput micro-drug library modeling module is configured to form drugs in a micro-tube, wherein the drugs are arranged in an ordered array of long micro-droplets and the micro-droplets are separated by oil to obtain a micro-drug library, and the microfluidic high-throughput drug screening module is configured to screen the drug micro-droplets sequentially output from the micro-tube in the micro-drug library.
Referring to fig. 1, the cell culture substrate is physically adhered to the bottom of the microfluidic chip, and the cell culture substrate is not exchanged with the microfluidic control system or the microfluidic chip.
Referring to fig. 1, the microfluidic chip is bonded under the microfluidic control system in a thermal-sealed chemical bond linkage manner, and is bonded on the cell culture substrate in a physical bonding manner; the diameter of a micro-well array in the micro-fluidic chip is 150-800 μm, and the height is 100-300 μm;
the device adopting the cell culture substrate, the microfluidic chip and the microfluidic control system has the advantages of simple structure, convenient and controllable operation, and overcomes the defect that the size of the organoid is difficult to be uniform due to the uncontrollable property in the operation process, and the problem of inconsistent number of organoids in each hole due to the uniform micropore array with consistent number in each microfluidic chip.
Referring to fig. 5, the hydrogel layer is composed of a natural hydrogel material and/or a synthetic hydrogel material;
the hydrogel is a 3D structure, and is preferably any one or more of a spheroid, a filament, a prism, a column, a block, a sheet, a capsule, a tube, a network, and a braid.
Referring to fig. 1 and 5, the synthetic hydrogel material includes one or more of polypropylene, polystyrene, polyacrylamide, polylactide, polyglycolide, polylactic acid, polylactic-co-glycolic acid, polyhydroxy acid, polylactic-co-alkyd, polydimethylsiloxane, polyanhydride, polyacid ester, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate, polyethylene oxide, or the like.
Referring to fig. 1 and 5, the natural hydrogel material includes one or more of gelatin or a derivative thereof, alginate or a derivative thereof, cellulose or a derivative thereof, agar, matrigel, collagen or a derivative thereof, amino acid or a derivative thereof, glycoprotein and a derivative thereof, hyaluronic acid or a derivative thereof, chitosan or a derivative thereof, layer-connecting protein, fibronectin, fibrin or a derivative thereof, silk fibroin or a derivative thereof, vitronectin, osteopontin, peptide fragment hydrogel, and DNA hydrogel.
The working principle of the method is as follows: firstly, cutting a tissue sample of an organoid into tissue blocks suitable for mechanical grinding, grinding the tissue blocks by a mechanical grinding method, performing first-stage filtration through a first-stage filter screen with the aperture of 80-200 μm to form first-stage filtrate, performing second-stage filtration on the first-stage filtrate through a second-stage filter screen with the aperture of 30-60 μm to obtain solids on the second-stage filter screen to obtain organoids, respectively introducing matrix hydrogel containing organoid cells and fluorine oil into a three-way device to obtain organoid spheres, and adhering a cell culture substrate below the microfluidic chip in a physical adhesion manner, the cell culture substrate and the microfluidic control system or the microfluidic chip are not subjected to material exchange, and a mechanical method can obviously enrich tumor cells, remove normal cells and obtain tumor organoids with higher purity.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
Claims (9)
1. A preparation method of organoid structure based on microfluidic technology high-throughput culture comprises a cell culture substrate, a microfluidic chip, a microfluidic control system, a high-throughput micro-drug library modeling module and a microfluidic high-throughput drug screening module, and is characterized by comprising the following steps:
step 1: obtaining organoid tissue samples and cutting them into tissue pieces suitable for mechanical grinding;
step 2: grinding the tissue block by a mechanical grinding method, and performing first-stage filtration by a first-stage filter screen with the aperture of 80-200 μm to form first-stage filtrate;
and step 3: performing second-stage filtration on the first-stage filtrate in a second-stage filter screen with the aperture of 30-60 mu m to obtain solids on the second-stage filter screen, and thus obtaining the organoid;
and 4, step 4: respectively introducing the matrix hydrogel containing organoid cells and the fluorine oil into a three-way device to obtain organoid spheres;
the flow rate of the cell-containing matrix hydrogel is 10-40 mu L/min; the flow rate of the fluorine oil is 40-55 mu L/min;
the cell culture substrate is adhered below the microfluidic chip and used for placing the microfluidic chip;
the microfluidic chip is adhered below the microfluidic control system and on the cell culture substrate, and a microwell array in the microfluidic chip is communicated with the microfluidic control system and used for exchanging substances in a pipeline of the microfluidic control system;
the microfluidic control system is adhered to the microfluidic chip, a microfluidic pipeline in the microfluidic control system is communicated with the microfluidic chip, the microfluidic control system controls microfluid while exchanging substances, and the output end of the microfluidic control system is connected with a 3D printing platform.
2. The method for preparing organoid structures for high throughput culture based on microfluidic technology according to claim 1, wherein the microfluidics is controlled while performing the material exchange, comprising: the filling of cells or tissues, the filling and replacement of culture media, the inflow and discharge of drugs during drug testing, and the control of microfluid required by experiments are performed.
3. The method for preparing organoid structure based on microfluidic high-throughput culture according to claim 2, wherein the organoid is wrapped in matrigel and cultured in organoid culture medium, the organoid culture medium comprises hydrogel layer and aqueous two-phase system, the aqueous two-phase system is prepared by: selecting a double aqueous phase system as a combination of PEG-dextran; the molecular weight range of PEG is as follows: 1000-: 2.5 to 50 percent; dextran molecular weight range: 70k-500kDa, concentration range: 2.5 to 20 percent.
4. The method for preparing a organoid structure through high-throughput culture based on microfluidic technology according to claim 2, wherein the high-throughput micro-drug library modeling module is configured to form drugs in a micro-tube with long droplets arranged in order and separated by oil, so as to obtain a micro-drug library, and the microfluidic high-throughput drug screening module is configured to perform drug screening on drug droplets in the micro-drug library sequentially output through the micro-tube.
5. The method for preparing organoid structures for high throughput culture based on microfluidic technology of claim 4, wherein the cell culture substrate is physically adhered under the microfluidic chip, and the cell culture substrate is not exchanged with the microfluidic control system or the microfluidic chip.
6. The method for preparing organoid structure with high throughput culture based on microfluidics technology of claim 5, wherein the microfluidic chip is bonded under the microfluidic control system in a heat-sealed chemical bonding linkage manner, and is bonded on the cell culture substrate in a physical bonding manner; the diameter of the micro-well array in the micro-fluidic chip is 150-800 μm, and the height is 100-300 μm.
7. The method for preparing organoid structures for high throughput culture based on microfluidics technology of claim 3, wherein the hydrogel layer comprises of natural hydrogel material and synthetic hydrogel material.
8. The method of claim 7, wherein the synthetic hydrogel material comprises one or more of polypropylene, polystyrene, polyacrylamide, polylactide, polyglycolide, polylactic acid-glycolic acid copolymer, polyhydroxy acid, polylactic acid-alkyd copolymer, polydimethylsiloxane, polyanhydride, polyacid ester, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate, polyethylene oxide, etc.
9. The method for preparing organoid structures for high-throughput culture based on microfluidic technology according to claim 8, wherein the natural hydrogel material comprises one or more of gelatin or its derivatives, alginate or its derivatives, cellulose or its derivatives, agar, matrigel, collagen or its derivatives, amino acids or its derivatives, glycoproteins and their derivatives, hyaluronic acid or its derivatives, chitosan or its derivatives, layer-connecting proteins, fibronectin, fibrin or its derivatives, silk fibroin or its derivatives, vitronectin, osteopontin, peptide fragment hydrogel, and DNA hydrogel.
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