CN117327300B - Hydrogel and preparation method thereof, organoid and culture method thereof, and drug detection method - Google Patents
Hydrogel and preparation method thereof, organoid and culture method thereof, and drug detection method Download PDFInfo
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Abstract
本发明提出一种水凝胶及其制备方法、类器官及其培养方法、药物检测的方法，属于生物医药技术领域。制备方法包括：获得GelMA‑胶原混合溶液；所述GelMA‑胶原混合溶液为中性溶液，所述GelMA‑胶原混合溶液包含GelMA、LAP光引发剂，I型胶原、氯化三(2,2'‑联吡啶)钌(II)六水合物光引发剂以及过硫酸钠光引发剂；所述GelMA‑胶原混合溶液经光固化形成具有交错互穿的第一层交联网络和第二层交联网络的水凝胶。本发明的制备方法简单，原料易获得，制备成本较低，且获得的水凝胶成分明确，机械强度较高且可调节，生物相容性好以及可满足不同类型类器官的培养需求与监控细胞生长状态需求。
The present invention proposes a hydrogel and a preparation method thereof, an organoid and a culture method thereof, and a method for drug detection, and belongs to the field of biomedical technology. The preparation method includes: obtaining a GelMA-collagen mixed solution; the GelMA-collagen mixed solution is a neutral solution, and the GelMA-collagen mixed solution contains GelMA, LAP photoinitiator, type I collagen, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator and sodium persulfate photoinitiator; the GelMA-collagen mixed solution is photocured to form a hydrogel having a first layer cross-linked network and a second layer cross-linked network interlaced and interpenetrating. The preparation method of the present invention is simple, the raw materials are easy to obtain, the preparation cost is low, and the obtained hydrogel has clear components, high and adjustable mechanical strength, good biocompatibility, and can meet the culture requirements of different types of organoids and the requirements for monitoring cell growth status.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to hydrogel, a preparation method thereof, organoids, a culture method thereof and a medicine detection method.
Background
Organoids are self-organizing cellular structures grown from Pluripotent Stem Cells (PSCs) or multipotent tissue resident Adult Stem Cells (ASCs), are valuable models for studying organ development and human disease, and provide valuable tissue sources for various biomedical applications such as drug screening and toxicology studies.
Organoid culture typically relies on the use of Basal Membrane Extracts (BMEs) of tumor origin. BME hydrogels consist of a heterogeneous mixture of extracellular matrix (ECM), proteoglycans, and growth factors of mouse sarcomas. However, BME hydrogels have weak mechanical properties, are difficult to adapt to various unique tumor microenvironments, and the heterogeneity of BME components limits precise control over their physical and biochemical characteristics, and the variability between heterogeneous components and batches greatly limits clinical applications of cells grown in such matrices, such as regenerative medicine and high-content screening.
Matrigel is currently applied to organoid culture, and can better meet the growth requirements of various organoids, however, because Matrigel components are not clear and are unstable among batches, the Matrigel greatly limits the use of the Matrigel in large-scale and standardized organoid culture and organoid drug screening drug sensitivity.
The synthetic hydrogel is used as a matrix material for supporting tissue regeneration, is used as a carrier for cell or drug delivery, or is used for cell culture, and most of the synthetic hydrogels proposed at present are single-component hydrogels, such as pure collagen, pure gelatin, poly (isonitrile) Polypeptide (PIC), modified product hydrogels thereof or polyethylene glycol (PEG) hydrogels, and the like, and have the problems of low mechanical strength, insufficient cell growth sites, poor biocompatibility, difficulty in meeting the requirement of being convenient for monitoring the growth state of cells, and the like. Synthetic hydrogels composed of multiple components are also proposed at present, however, the preparation process of the components is relatively complex, the biocompatibility is poor, and the mechanical strength is single. Organoids have higher requirements for mechanical properties and biocompatibility of the culture carrier when cultured, and thus present challenges in simulating tissue-like three-dimensional structures and constructing microenvironments using synthetic hydrogels.
Disclosure of Invention
The invention aims at solving at least one of the technical problems in the prior art and provides hydrogel, a preparation method thereof, organoids, a culture method thereof and a drug detection method.
In one aspect of the present invention, a method for preparing a hydrogel is provided, the method comprising:
obtaining a GelMA-collagen mixed solution;
The GelMA-collagen mixed solution is a neutral solution, the GelMA-collagen mixed solution comprises GelMA and LAP photoinitiators, type I collagen, tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photoinitiators and sodium persulfate photoinitiators,
In the GelMA-collagen mixed solution, the concentration of the tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate ranges from 0.0005 to 0.005mol/L;
The concentration range of the sodium persulfate photoinitiator is 0.005-0.05mol/L;
The concentration range of the LAP photoinitiator is 0.05% -0.5%;
the concentration range of the GelMA is 0.5% -20%;
the concentration range of the type I collagen is 0.05-1%;
the GelMA-collagen mixed solution is subjected to photo-curing to form hydrogel with a first layer of cross-linked network and a second layer of cross-linked network which are staggered and interpenetrating, wherein the first layer of cross-linked network is formed by GelMA through photo-curing under the action of LAP photo-initiator;
The second layer of crosslinked network is formed by photocuring type I collagen under the action of a tri (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photoinitiator and a sodium persulfate photoinitiator.
Optionally, the photo-curing time ranges from 0.5 to 5 minutes.
In another aspect of the present invention, a hydrogel is provided, which is prepared by the preparation method described above.
In another aspect of the invention, a hydrogel is provided comprising a first layer of crosslinked network, and a second layer of crosslinked network interpenetrating the first layer of crosslinked network, wherein,
The first layer of crosslinked network is formed by GelMA through photo-curing under the action of LAP photoinitiator;
The second layer of crosslinking network is formed by photocuring type I collagen under the action of a tri (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photoinitiator and a sodium persulfate photoinitiator;
in GelMA and LAP photoinitiator, a GelMA-collagen mixed solution formed by type I collagen, tris (2, 2 '-bipyridine) ruthenium (II) chloride hexahydrate photoinitiator and sodium persulfate photoinitiator, wherein the concentration of the tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate ranges from 0.0005 mol/L to 0.005mol/L;
The concentration range of the sodium persulfate photoinitiator is 0.005-0.05mol/L;
The concentration range of the LAP photoinitiator is 0.05% -0.5%;
the concentration range of the GelMA is 0.5% -20%;
the concentration range of the type I collagen is 0.05-1%.
In another aspect of the present invention, there is provided a method of organoid culture, comprising:
forming a cell mass solution of the organoid to be cultured, wherein the cell mass in the cell mass solution is obtained by digestion and separation of tumor tissues or non-tumor tissues or by digestion and separation of the organoid to be passaged or the non-tumor organoid;
The preparation method comprises the steps of obtaining a GelMA-collagen mixed solution, wherein the GelMA-collagen mixed solution is a neutral solution, the GelMA-collagen mixed solution comprises GelMA and LAP photoinitiator, type I collagen, tris (2, 2 '-bipyridine) ruthenium (II) hexahydrate photoinitiator and sodium persulfate photoinitiator, the GelMA-collagen mixed solution is a neutral solution, the substitution degree of GelMA in the GelMA-collagen mixed solution is 30% -95%, the concentration range of GelMA is 0.5% -20%, the concentration range of LAP photoinitiator is 0.05% -0.5%, the concentration range of type I collagen in the GelMA-collagen mixed solution is 0.05% -1%, the concentration range of tris (2, 2' -bipyridine) ruthenium (II) hexahydrate is 0.0005-0.005mol/L, and the concentration range of sodium persulfate photoinitiator is 0.005-0.05mol/L;
Mixing the GelMA-collagen mixed solution with the cell mass solution, adding a proper amount of mixed solution into a culture cavity or a culture hole for culturing the organoid, and performing photo-curing to form organoid gel with a first layer of cross-linked network and a second layer of cross-linked network which are staggered and interpenetrating, wherein the first layer of cross-linked network is formed by photo-curing of the GelMA under the action of the LAP photo-initiator, and the second layer of cross-linked network is formed by photo-curing of the type I collagen under the action of the tri (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photo-initiator and the sodium persulfate photo-initiator;
and adding a culture medium into a culture cavity or a culture hole containing the organoid gel, and culturing to obtain the tumor organoid or the non-tumor organoid.
Optionally, when the organoid is any of liver, colorectal, pancreatic and pancreatic cancers, the concentration of GelMA in the GelMA-collagen mixed solution ranges from 0.5% to 10%, and the concentration of type I collagen ranges from 0.05% to 0.5%.
Optionally, when the organoid is any one of liver cancer and colorectal cancer, the concentration range of GelMA in the GelMA-collagen mixed solution is 10% -20%, and the concentration range of type I collagen is 0.5% -1%.
In another aspect of the present invention, a method of culturing a lung or lung cancer organoid is presented, the method comprising:
forming a lung or lung cancer cell mass solution, wherein the cell mass in the lung or lung cancer cell mass solution is obtained by digestion and separation of lung or lung cancer tissues or by digestion and separation of lung or lung cancer organoids to be passaged;
The preparation method comprises the steps of obtaining a GelMA-collagen mixed solution, wherein the GelMA-collagen mixed solution comprises GelMA and LAP photoinitiator, the type I collagen, tris (2, 2 '-bipyridine) ruthenium (II) hexahydrate photoinitiator and sodium persulfate photoinitiator, the GelMA-collagen mixed solution is a neutral solution, the substitution degree of the GelMA in the GelMA-collagen mixed solution is 30% -95%, the concentration range of the GelMA is 0.5% -20%, the concentration range of the LAP photoinitiator is 0.05% -0.5%, the concentration range of the type I collagen in the GelMA-collagen mixed solution is 0.05% -1%, the concentration range of the tris (2, 2' -bipyridine) ruthenium (II) hexahydrate is 0.0005-0.005mol/L, and the concentration range of the sodium persulfate photoinitiator is 0.005-0.05mol/L;
Mixing the GelMA-collagen mixed solution with a lung or lung cancer cell mass solution, adding a proper amount of mixed solution into a culture cavity or a culture hole, and performing photo-curing to form lung or lung cancer organoid gel with a first layer of cross-linked network and a second layer of cross-linked network which are staggered and interpenetrating, wherein the first layer of cross-linked network is formed by photo-curing the GelMA under the action of an LAP photo-initiator, and the second layer of cross-linked network is formed by photo-curing the type I collagen under the action of a tris (2, 2' -bipyridine) ruthenium (II) hexahydrate photo-initiator and a sodium persulfate photo-initiator;
And adding a lung or lung cancer organoid culture medium into a culture cavity or a culture hole containing the lung or lung cancer organoid gel, and culturing to obtain the lung or lung cancer organoid.
In another aspect of the present invention, there is provided an organoid which is cultured by the culture method described above.
In another aspect of the invention, a method for drug detection using the organoids described above is presented, the method comprising:
introducing a drug to be tested into the organoid;
and obtaining the action result of the drug to be tested on the organoid.
The invention provides hydrogel, a preparation method thereof, an organoid, a culture method thereof and a drug detection method, wherein the preparation method comprises the steps of obtaining a GelMA-collagen mixed solution, wherein the GelMA-collagen mixed solution is a neutral solution, the GelMA-collagen mixed solution comprises GelMA and LAP photoinitiators, type I collagen, tris (2, 2' -bipyridine) ruthenium (II) hexahydrate photoinitiator and sodium persulfate photoinitiator, and the GelMA-collagen mixed solution is subjected to photocuring to form interpenetrating network hydrogel. The preparation method disclosed by the invention is simple, raw materials are easy to obtain, the preparation cost is lower, the obtained hydrogel is clear in component, high in mechanical strength and adjustable, the biocompatibility is good, and the culture requirements of different types of organoid tissues and the requirements of monitoring the growth state of cells can be met.
Drawings
FIG. 1 is a flow chart of a method for preparing a hydrogel according to an embodiment of the invention;
FIG. 2 is a schematic structural view of a hydrogel according to another embodiment of the present invention;
FIG. 3 is a flow chart of a method of organoid culture according to another embodiment of the invention;
FIG. 4 is a flow chart of a method of organ-like culture of lung or lung cancer according to another embodiment of the invention;
FIG. 5 is a flow chart of a method for drug detection using organoids in accordance with another embodiment of the present invention;
FIG. 6 is a scanning electron microscope view of the homemade hydrogel of example 1 and Matrigel of the control group, wherein A, B of FIG. 6 is an electron microscope view of Matrigel of the control group at 1000x,5000x, respectively, and C, D of FIG. 6 is an electron microscope view of homemade hydrogel of example 1 at 1000x,5000x, respectively;
FIG. 7 is a microscopic view of a self-made hydrogel of example 2 of the present invention and a control Matrigel for culturing pancreatic cancer organoids;
FIG. 8 is a microscopic view of a self-made hydrogel of example 3 of the present invention and a control Matrigel for culturing colorectal cancer organoids;
FIG. 9 is a microscopic view of a self-made hydrogel and a control Matrigel of example 4 of the present invention used for culturing a mouse lung organ;
FIG. 10 is a microscopic view of the liver organ of a cultured mouse using the homemade hydrogel of example 5 of the present invention and a control Matrigel.
Detailed Description
The present invention will be described in further detail below with reference to the drawings and detailed description for the purpose of better understanding of the technical solution of the present invention to those skilled in the art. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without creative efforts, based on the described embodiments of the present invention belong to the protection scope of the present invention.
As shown in FIG. 1, in one aspect of the present invention, a method S100 for preparing a hydrogel for organoid culture is provided, comprising steps S110 to S120:
s110, obtaining a GelMA-collagen mixed solution, wherein the GelMA-collagen mixed solution is a neutral solution, and comprises GelMA and LAP photoinitiators, type I collagen, tris (2, 2' -bipyridine) ruthenium (II) hexahydrate photoinitiator and sodium persulfate photoinitiator.
In the present embodiment, the photo-curing agent corresponding to the GelMA component is LAP photo-initiator, the photo-curing agent corresponding to the type I collagen is tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photo-initiator and sodium persulfate photo-initiator, and the mixing order of the components is not particularly limited, so long as the components and the corresponding photo-initiators are mixed before photo-curing. That is, the GelMA-collagen mixed solution comprises a GelMA solution and a neutral collagen solution, wherein the GelMA and LAP photoinitiators are mixed to form the GelMA solution, the type I collagen, the tris (2, 2' -bipyridine) ruthenium (II) hexahydrate photoinitiator and the sodium persulfate are mixed to form the neutral collagen solution, and the neutral collagen solution and the GelMA solution are mixed to form the GelMA-collagen mixed solution, or the GelMA solution is formed after the neutral collagen solution is formed, and the GelMA-collagen mixed solution is formed after the GelMA-collagen mixed solution is mixed. Of course, in addition to the above sequence, two solutions may be formed simultaneously, or one of the solutions may be formed in synchronization with the mixing process of the two solutions. For example, adding the type I collagen solution, the tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photoinitiator and the sodium persulfate photoinitiator to the GelMA solution to obtain a GelMA-collagen mixed solution, or adding the GelMA solution and the LAP photoinitiator to the neutral collagen solution to obtain the GelMA-collagen mixed solution.
Illustratively, in some preferred embodiments, the GelMA-collagen mixed solution forming process comprises the specific steps of dissolving GelMA in water, adding a phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite (LAP) photoinitiator, mixing uniformly in the dark to obtain a GelMA solution, adding type I collagen to the GelMA solution, adding a tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photoinitiator and a sodium persulfate photoinitiator, mixing uniformly in the dark to obtain a GelMA-collagen mixed solution.
In other preferred embodiments, the GelMA-collagen mixed solution forming process comprises the specific steps of dissolving type I collagen in acetic acid solution, adding NaOH to adjust pH to neutrality, adding an initiator of tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photoinitiator and a sodium persulfate photoinitiator, uniformly mixing in a dark place to obtain a neutral collagen solution, adding GelMA into the neutral collagen solution, adding a certain amount of LAP photoinitiator, and uniformly mixing in a dark place to obtain the GelMA-collagen mixed solution.
In some preferred embodiments, the type I collagen is naturally extracted animal collagen or recombinant collagen prepared by a microbiological method.
In the GelMA-collagen mixed solution, the GelMA substitution degree is 30% -95%, the GelMA concentration is 0.5% -20%, the LAP photoinitiator concentration is 0.05% -0.5%, the type I collagen concentration is 0.05% -1%, the tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photoinitiator concentration is 0.0005% -0.005 mol/L, and the sodium persulfate photoinitiator concentration is 0.005% -0.05 mol/L. Wherein the unit of the concentration ranges of LAP, gelMA and type I collagen is mass-volume ratio.
In some preferred embodiments, the GelMA concentration may preferably be 0.5% -10%, and the type I collagen concentration may preferably be 0.05% -0.5%, and the mechanical strength of the hydrogel formed in the preferred concentration range is matched to organs or tissues having a low young's modulus, such as liver, colorectal, pancreatic and pancreatic cancers having young's modulus of several kilopascals.
In other preferred embodiments, the concentration of GelMA may be preferably 10% -20%, the concentration of type I collagen is preferably 0.5% -1%, and the mechanical strength of the hydrogel formed in the above preferred concentration range is matched with that of an organ or tissue having a higher young's modulus, for example, liver cancer, colorectal cancer, etc. having a young's modulus of several tens kilopascals.
S120, photo-curing the GelMA-collagen mixed solution for 0.5-5 minutes to form the hydrogel of the interpenetrating network.
Specifically, in the GelMA-collagen mixed solution, the GelMA solution is subjected to photo-curing to form a first layer of crosslinked network, the neutral collagen solution is subjected to photo-curing to form a second layer of crosslinked network, and the first layer of crosslinked network and the second layer of crosslinked network are interlaced and interpenetrating.
When the above GelMA-collagen mixed solution is used for organoid culture, preferably, the mixed solution is filtered by a 0.22 micrometer filter membrane to obtain a sterile GelMA-collagen mixed solution, and then the mixed solution is mixed with a organoid cell mass solution to be cultured subsequently, and the organoid cell mass solution is subjected to photocrosslinking to form hydrogel with cells.
When the hydrogel of the embodiment is used for organoid culture, the type I collagen in the hydrogel can be subjected to temperature-sensitive solidification according to requirements under the organoid culture condition, so that the crosslinking degree is further increased, the mechanical strength of the hydrogel is improved, a heating step is not required to be independently arranged, the complexity and the preparation cost of the preparation process are greatly reduced, and the time of the preparation process is shortened.
The preparation process of the hydrogel is simple, the interpenetrating network structure is formed by means of dual crosslinking photo-curing polymerization based on the fact that two components are respectively provided with the photoinitiator, the crosslinking degree is effectively improved, the mechanical strength of the hydrogel is further effectively improved, the hydrogel with different mechanical strengths is formed based on the fact that the concentration of each component is adjusted in the preparation process, and the type I collagen is photo-cured into gel under the action of the tri (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photoinitiator and the sodium persulfate photoinitiator to form transparent hydrogel, so that the growth state of organoid tissues can be observed conveniently.
In another aspect of the present invention, a hydrogel for organoid culture is provided, the hydrogel is prepared by the method described above, and the specific preparation process is referred to above, and is not described herein.
As shown in fig. 2, the hydrogel comprises a first layer of crosslinked network and a second layer of crosslinked network which is intersected and interpenetrating with the first layer of crosslinked network, wherein the first layer of crosslinked network is formed by GelMA through photocuring under the action of LAP photoinitiator, and the second layer of crosslinked network is formed by photocuring type I collagen under the action of tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photoinitiator and sodium persulfate photoinitiator.
In the embodiment, under the condition of light irradiation, gelMA double bond polymerization reaction forms a first layer of cross-linked network, amino acid residue on type I collagen is oxidized and coupled to form a second layer of cross-linked network, and the two layers of network structures are connected with amino groups on partial double-bonded GelMA through carboxyl groups on collagen to form an interpenetrating network, so that the interpenetrating network has stronger mechanical property.
In the preparation process of the hydrogel, gelMA, LAP photoinitiator, type I collagen, tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photoinitiator and sodium persulfate photoinitiator are mixed to form a GelMA-collagen mixed solution, and the GelMA-collagen mixed solution is subjected to photo-curing to form a two-layer interpenetrating crosslinked network. In the GelMA-collagen mixed solution, the concentration range of the tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate is 0.0005-0.005mol/L, the concentration range of the sodium persulfate photoinitiator is 0.005-0.05mol/L, the concentration range of the LAP photoinitiator is 0.05-0.5%, the concentration range of the GelMA is 0.5-20%, and the concentration range of the type I collagen is 0.05-1%.
In the embodiment, the hydrogel has an interpenetrating network structure and a porous structure, has compact pore canal and rich pore canal layers, is favorable for supplementing active sites required by specific cell growth and supporting the three-dimensional growth of cells, has definite components, only comprises GelMA and I-type collagen, is in a transparent state, has the advantages of good biological activity, high mechanical strength, adjustability and the like, can meet the culture requirements of different types of organoid tissues, and solves the problems that the existing hydrogel adopting collagen is difficult to simultaneously meet the requirements of high-definition observation and measurement of the growth state and the drug testing state of the organoid tissues and the requirement of the growth activity of the organoid tissues.
As shown in FIG. 3, another aspect of the present invention provides an organoid culture method S200, comprising the following steps S210 to S230:
S210, forming a cell mass solution of the organoid to be cultured, wherein the cell mass in the cell mass solution is obtained by digestion and separation of primary tumor tissues or non-tumor tissues or by digestion and separation of the organoid to be passaged or the non-tumor organoid.
In this embodiment, the organoids to be cultured may include non-tumor organoids, for example, pancreas, colorectal/small intestine, liver, and the like of mice, and may also be tumor organoids, for example, organoids such as liver cancer and colorectal cancer. That is, the pancreatic cancer tissue, colorectal cancer tissue, liver cancer tissue, etc. are digested to obtain the corresponding cancer tissue, or the mouse pancreas, colorectal/small intestine, liver, etc. are digested to obtain the normal tissue cell mass precipitate, and then the corresponding cancer tissue or the cell mass precipitate is resuspended to obtain the cell mass solution.
Specifically, the organoids which are subjected to primary extraction or passage are selected, PBS which is pre-cooled in advance is added into a culture pore plate, 1mL of PBS is added into each pore, the mixture is collected into a centrifuge tube, the mixture is refrigerated at 4 ℃ for 15min, centrifugation (1000 rpm,5 min) is carried out, supernatant is removed to obtain organoid sediment, tryple E is added for enzymolysis for 1min, centrifugation (1000 rpm,5 min) is carried out, supernatant is removed to obtain sediment, PBS is added for centrifugation (1000 rpm,5 min) again, redundant enzyme solution is removed to obtain sediment, and the sediment is resuspended to obtain cell mass solution.
S220, obtaining a GelMA-collagen mixed solution, wherein the GelMA-collagen mixed solution comprises GelMA and LAP photoinitiator, type I collagen, tris (2, 2 '-bipyridine) ruthenium (II) hexahydrate photoinitiator and sodium persulfate photoinitiator, wherein the substitution degree of GelMA in the GelMA-collagen mixed solution is 30-95%, the concentration range of GelMA is 0.5-20%, the concentration range of LAP photoinitiator is 0.05-0.5%, the concentration range of type I collagen in the GelMA-collagen mixed solution is 0.05-1%, the concentration range of tris (2, 2' -bipyridine) ruthenium (II) hexahydrate is 0.0005-0.005mol/L, and the concentration range of sodium persulfate photoinitiator is 0.005-0.05 mol/L.
S230, mixing the GelMA-collagen mixed solution with the cell mass solution, adding a proper amount of the mixed solution into a culture cavity or a culture hole for culturing the organoid, and performing photo-curing to form the organoid gel.
Specifically, uniformly mixing a GelMA-collagen mixed solution and a cell mass precipitation solution in a ratio of 85:15 (v/v) to form a cell-gel mixed solution, adding 5-30 microliters of the cell-gel mixed solution into a culture hole or a culture cavity according to 50-500 organoids per hole, and then irradiating for 0.5-5min under blue light to crosslink and gel to form organoid gel containing cells.
In the crosslinking and gelling process, gelMA is subjected to photocuring under the action of LAP photoinitiator in the mixed solution of GelMA-collagen mixed solution and cell mass solution to form a first layer of crosslinking network, and type I collagen is subjected to photocuring under the action of tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photoinitiator and sodium persulfate photoinitiator to form a second layer of crosslinking network, wherein the first crosslinking network and the second crosslinking network are interlaced and interpenetrating, namely, the interpenetrating network organogel containing cells is formed.
S240, adding a culture medium into a culture cavity or a culture hole containing the organoid gel, and culturing to obtain the tumor organoid or the non-tumor organoid.
Specifically, the corresponding organoid culture medium is added into the culture hole or the culture cavity, and the organoid culture medium is placed into an incubator with 37 ℃ and 5% CO 2 and 95% humidity for culture, and organoid amplification culture is carried out.
Further, digesting the amplified organoids to obtain organoid cell clusters, re-suspending the organoid cell clusters by using a GelMA-collagen mixed solution, adding the organoids into a culture cavity or a culture hole according to the quantity of 10-50 organoids per hole, performing gel formation by blue light irradiation for 0.5-5min, adding corresponding organoid culture mediums, placing the organoids in a culture box with 37 ℃ and 5% CO 2 and 95% humidity for culture, replacing the culture mediums containing the medicine to be detected after 1-3 days, and performing cell activity detection after 5-10 days of culture.
When organoids are cultured at the above-mentioned culture temperature, the type I collagen in the hydrogel may be further subjected to temperature-sensitive crosslinking as needed, thereby improving the mechanical strength of the hydrogel.
It should be further noted that, in this embodiment, for organoids with different young's modulus, hydrogels with different mechanical strength can be formed by adjusting the concentration of each component during the preparation process of the hydrogel, so as to match organoids with different young's modulus.
Illustratively, when the organoid is a low Young's modulus (several kilopascals) organoid, e.g., liver, colorectal, pancreatic/pancreatic cancer, etc., the organoid gel is prepared as follows:
S1, dissolving GelMA in water, adding an initiator phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite (LAP), and uniformly mixing in a dark place to obtain a GelMA mixed solution; adding type I collagen into GelMA mixed solution, adding an initiator of tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate and sodium persulfate, uniformly mixing in a dark place, and filtering by a 0.22 micrometer filter membrane to obtain sterile GelMA-collagen mixed solution;
In the step S1, the substitution degree of GelMA is 30% -95%, the concentration of GelMA is 0.5% -10%, the concentration of an initiator LAP is 0.05% -0.5%, the concentration of collagen is 0.05% -0.5%, the concentration of an initiator tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate is 0.0005-0.005mol/L, and the concentration of sodium persulfate is 0.005-0.05mol/L in the GelMA-collagen mixed solution.
S2, selecting primary extraction or organoids to be passaged, adding pre-chilled PBS into a culture pore plate, adding 1mL of PBS into each pore, collecting in a centrifuge tube, refrigerating at 4 ℃ for 15min, centrifuging (1000 rpm,5 min), removing the supernatant to obtain organoid precipitate, adding Tryple E for enzymolysis for 1min, centrifuging (1000 rpm,5 min), removing the supernatant to obtain precipitate, adding PBS for centrifuging (1000 rpm,5 min) again, removing redundant enzyme solution to obtain precipitate, and re-suspending to obtain cell mass solution;
s3, uniformly mixing the sterile GelMA-collagen mixed solution and the cell mass solution according to the proportion of 85:15 (v/v), dripping glue into a culture pore plate, obtaining a cell-gel mixed solution by 5-30 microliters per pore, crosslinking for 0.5-5 minutes under blue light irradiation, obtaining organoid gel, and adding the organoid gel into a corresponding culture medium for culture observation.
Illustratively, when the organoid is one having a high Young's modulus (several tens of kilopascals), for example, liver cancer, colorectal cancer, etc., the organoid gel is prepared as follows:
S1, dissolving GelMA in water, adding an initiator phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite (LAP), and uniformly mixing in a dark place to obtain a GelMA mixed solution; adding type I collagen into GelMA mixed solution, adding an initiator of tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate and sodium persulfate, uniformly mixing in a dark place, and filtering by a 0.22 micrometer filter membrane to obtain sterile GelMA-collagen mixed solution;
In the step S1, the substitution degree of GelMA is 30% -95%, the concentration of GelMA is 10% -20%, the concentration of an initiator LAP is 0.05% -0.5%, the concentration of collagen is 0.5% -1%, the concentration of an initiator tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate is 0.0005-0.005mol/L, and the concentration of sodium persulfate is 0.005-0.05mol/L in the GelMA mixed solution.
S2, selecting primary extraction or organoids to be passaged, adding pre-chilled PBS into a culture pore plate, adding 1mL of PBS into each pore, collecting in a centrifuge tube, refrigerating at 4 ℃ for 15min, centrifuging (1000 rpm,5 min), removing the supernatant to obtain organoid precipitate, adding Tryple E for enzymolysis for 1min, centrifuging (1000 rpm,5 min), removing the supernatant to obtain precipitate, adding PBS for centrifuging (1000 rpm,5 min) again, removing redundant enzyme solution to obtain precipitate, and re-suspending to obtain cell mass solution;
s3, uniformly mixing the sterile GelMA-collagen mixed solution and the cell mass solution according to the proportion of 85:15 (v/v), dripping glue into a culture pore plate, obtaining a cell-gel mixed solution by 5-30 microliters per pore, crosslinking for 0.5-5 minutes under blue light irradiation, obtaining organoid gel, and adding the organoid gel into a corresponding culture medium for culture observation.
In the embodiment, when the interpenetrating network hydrogel is used for culturing organoids, a reliable organoid in-vitro model is formed, the culture method is simple and easy to operate, and the hydrogel with different mechanical strengths is formed by adjusting the concentration of components in the hydrogel, so that the mechanical strength and the biological activity required by different organoids are met.
As shown in FIG. 4, in another aspect of the present invention, a method S300 for culturing a lung or lung cancer organoid is provided, comprising the following steps S310 to S340:
S310, forming a lung or lung cancer cell mass solution, wherein the cell mass in the lung or lung cancer cell mass solution is obtained by digestion and separation of lung or lung cancer tissues or by digestion and separation of lung or lung cancer organoids to be passaged;
s320, obtaining a GelMA-collagen mixed solution, wherein the GelMA-collagen mixed solution comprises GelMA and LAP photoinitiator, type I collagen, tris (2, 2 '-bipyridine) ruthenium (II) chloride hexahydrate photoinitiator and sodium persulfate photoinitiator, the GelMA-collagen mixed solution is a neutral solution, the substitution degree of the GelMA in the GelMA-collagen mixed solution is 30% -95%, the concentration range of the GelMA is 0.5% -20%, the concentration range of the LAP photoinitiator is 0.05% -0.5%, the concentration range of type I collagen in the GelMA-collagen mixed solution is 0.05% -1%, the concentration range of tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate is 0.0005-0.005mol/L, and the concentration range of the sodium persulfate photoinitiator is 0.005-0.05mol/L.
S330, mixing the GelMA-collagen mixed solution with the lung or lung cancer cell mass solution, taking a proper amount of mixed solution, adding the mixed solution into a culture cavity or a culture hole, and performing photo-curing to form the lung or lung cancer organoid gel.
S340, adding a lung or lung cancer organoid culture medium into a culture cavity or a culture hole containing lung or lung cancer organoid gel, and culturing to obtain the lung or lung cancer organoid.
Note that, in this embodiment, the process of culturing the lung or lung cancer organoids is the same as the other organoid culturing processes described above, and reference is made to the above description, and no further description is given here.
In another aspect of the present invention, an organoid is provided, and a plurality of organoids can be cultured by using the organoid culture method described above, and the detailed culture method is referred to above, and is not described herein.
Exemplary organoids include non-neoplastic organoids including pancreatic organoids, colorectal organoids, small intestine organoids, lung organoids, liver organoids, and neoplastic organoids including pancreatic cancer organoids, colorectal organoids, lung cancer organoids, liver cancer organoids.
In some preferred embodiments, the organoids include pancreatic, rectal, lung, liver organoids, etc., although other organoids may be cultured as desired and are not specifically described herein.
As shown in fig. 5, in another aspect of the present invention, a method S400 for detecting a drug by using an organoid is provided, which includes steps S410 to S420:
S410, introducing a drug to be tested into the organoid;
S420, obtaining the action result of the drug to be tested on the organoid.
In this embodiment, based on the above-described organoid in vitro model, the complexity of the human specific microenvironment, extracellular matrix, etc. can be simulated, and drug screening can be realized, and particularly effective drugs against cancers such as pancreatic cancer, rectal cancer, lung cancer, etc. can be screened out.
Illustratively, conventional drug susceptibility testing is performed on an orifice plate for a tumor organoid, comprising the steps of:
(1) Preparing a medicine-containing culture medium with proper concentration,
(2) The tumor cancer organoids are placed in culture wells in a non-dynamic culture environment, for example, in culture wells of a 96-well plate;
(4) Observing and recording the growth state of the tumor organoids, sucking out the culture medium in the culture holes, adding 100 mu L of the culture medium containing the liquid medicine into each hole, and placing the culture medium into a culture box with the temperature of 37 ℃ and the concentration of 5% CO2 for culture;
(4) Observing and recording the growth state of the tumor organoids on days 3-5;
(5) Activity assays were performed using CTG assay kit. Adding 100 mu L of CTG detection reagent into each hole, oscillating for 5min by using an oscillator, incubating for 25min at room temperature, and detecting by using a chemiluminescent enzyme-labeled instrument after incubation is finished.
Illustratively, conventional drug susceptibility testing of tumor organoids is performed under dynamic culture, comprising the steps of:
(1) Preparing a medicine-containing culture medium with proper concentration:
(2) The tumor organoids formed by culturing are placed in a culture cavity of a dynamic culture environment, for example, a chip with three communicated cavities is adopted, and the tumor organoids are placed in a middle cavity;
(3) The growth status of the tumor organoids was observed and recorded. The culture medium in the chip is sucked out, and the culture medium containing the liquid medicine is added. 50 mu L of culture medium is added into the holes on the left side and the right side of the chip, and 30 mu L of culture medium is added into the middle hole;
(4) After the medicine is added, the chip is put into a swinging perfusion instrument for 3 DEG/120 min for flow culture;
(5) Observing and recording the growth state of the tumor organoids on the 3 rd day to the 5 th day, and supplementing 30 mu L of drug-containing culture medium to the middle hole;
(6) Activity assays were performed using CTG assay kit. All the culture medium is discarded from the two side holes, 20 mu L of CTG diluent (1:1 dilution of CTG reagent stock solution and culture medium) is added, 50 mu L of CTG stock solution is added, the mixture is blown and evenly mixed, a shaker is used for shaking for 5min, the mixture is placed at room temperature for 25min, all the lysate is taken out, and the lysate is placed into a 96-well plate with completely opaque periphery and bottom. Detection was performed using a chemiluminescent microplate reader.
The method of preparation of hydrogels and organoid culture methods will be further described in connection with several specific examples:
Example 1
The embodiment provides a preparation method of GelMA-collagen interpenetrating network hydrogel, which comprises the following steps:
s1, dissolving GelMA with the substitution degree of 60% in water, adding an initiator phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite (LAP), and uniformly mixing in a dark place to obtain a GelMA mixed solution with the final concentration of 2% and the LAP concentration of 0.05%;
S2, adding type I collagen into the GelMA mixed solution, adding an initiator of tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate and sodium persulfate, and uniformly mixing in a dark place to obtain the GelMA-collagen mixed solution, wherein the concentration of the collagen is 0.2%, the concentration of the ruthenium (II) complex is 0.0005mol/L, and the concentration of the sodium persulfate is 0.005mol/L;
s3, crosslinking the GelMA-collagen mixed solution for 1min under blue light irradiation to obtain the GelMA-collagen interpenetrating network hydrogel, wherein the structure of the GelMA-collagen interpenetrating network hydrogel is shown in figure 2.
Further, the hydrogel obtained in this example 1 was compared with a commercial Matrigel matrix of a control group prepared by curing the commercial Matrigel at 37℃for 30 minutes, and both hydrogels obtained in this example were freeze-dried and observed by a scanning electron microscope. As shown in fig. 6, compared with matrigel of the control group, in the present embodiment, the porous structure of the hydrogel has dense pore channels and rich pore channel layers, which is beneficial to support the three-dimensional growth of cells, and the shape of the edge structure of the pore channels presents a non-smooth fiber shape, which effectively improves the viscoelasticity of the hydrogel, and is more beneficial to changing the morphology, precipitating the matrix and migrating cells by overcoming the mechanical limitation in the organoid culture process.
Example 2
The present example shows a method for preparing a hydrogel and a method for culturing pancreatic cancer organoids using the hydrogel, comprising:
S1, dissolving GelMA with the substitution degree of 60% in water, adding an initiator phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite (LAP), and uniformly mixing in a dark place to obtain a GelMA mixed solution with the final concentration of 10% and the LAP concentration of 0.1%;
S2, adding the type I collagen into the GelMA mixed solution, adding an initiator of tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate and sodium persulfate, uniformly mixing in a dark place, and filtering by a 0.22 micrometer filter membrane to obtain the sterile GelMA-collagen mixed solution. Wherein the concentration of collagen is 0.5%, the concentration of ruthenium (II) complex is 0.001mol/L, and the concentration of sodium persulfate is 0.01mol/L;
S3, selecting pancreatic cancer organoids to be passaged, adding pre-cooled PBS into a 24-well plate, adding 1mL of PBS into each well, collecting in a centrifuge tube, refrigerating at 4 ℃ for 15min, centrifuging (1000 rpm,5 min), removing supernatant to obtain organoid precipitates, adding Tryple E for enzymolysis for 1min, centrifuging (1000 rpm,5 min), removing supernatant to obtain precipitates, adding PBS for centrifuging (1000 rpm,5 min), removing redundant enzyme solution to obtain precipitates, and re-suspending the precipitates to form a cell mass solution.
S4, uniformly mixing the sterile GelMA-collagen mixed solution and the precipitated cell mass solution in a ratio of 85:15 (v/v), dripping a 24-pore plate, 30 microliters per pore, and curing for 15min in blue light to form gel.
Further, pancreatic cancer organoids obtained in example 2 were compared with pancreatic cancer organoids formed by commercial Matrigel of the control group (commercial Matrigel was uniformly mixed with the precipitated cell mass solution, 24 well plates were dropped, 30. Mu.l per well, solidified at 37℃for 30min to gel, and cultured in pancreatic cancer medium to obtain pancreatic cancer organoids). As shown in fig. 7, compared with the control group, the self-made hydrogel of the present embodiment has a better pancreatic cancer organoid status and shows a trend of increasing with time, which indicates that the hydrogel of the present embodiment satisfies the requirements for pancreatic cancer organoid culture and has the capability of three-dimensional cell culture organoids.
Example 3
The present example shows a method for preparing a hydrogel and a method for culturing colorectal cancer organoids using the same, comprising:
s1, dissolving GelMA with the substitution degree of 60% in water, adding an initiator phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite (LAP), and uniformly mixing in a dark place to obtain a GelMA mixed solution with the final concentration of 12% and the LAP concentration of 0.2%;
s2, adding the type I collagen into the GelMA mixed solution, adding an initiator of tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate and sodium persulfate, uniformly mixing in a dark place, and filtering by a 0.22 micrometer filter membrane to obtain the sterile GelMA-collagen mixed solution. Wherein the concentration of collagen is 0.6%, the concentration of ruthenium (II) complex is 0.001mol/L, and the concentration of sodium persulfate is 0.01mol/L;
S3, selecting a primary colorectal cancer organoid, adding pre-cooled PBS into a 24-hole plate, adding 1mL of PBS into each hole, collecting in a centrifuge tube, refrigerating at 4 ℃ for 15min, centrifuging (1000 rpm,5 min) to remove supernatant to obtain organoid precipitate, adding Tryple E for enzymolysis for 1min, centrifuging (1000 rpm,5 min) to remove supernatant to obtain precipitate, adding PBS for centrifuging (1000 rpm,5 min) again to remove redundant enzyme solution to obtain precipitate, and re-suspending the precipitate to form a cell mass solution.
S4, uniformly mixing the sterile GelMA-collagen mixed solution and the precipitated cell mass solution in a ratio of 85:15 (v/v), dripping a 24-pore plate, 30 microliters per pore, and curing for 15min in blue light to form gel.
Further, colorectal cancer organoids obtained in this example 3 were compared with colorectal cancer organoids formed by commercial Matrigel of the control group (commercial Matrigel was uniformly mixed with the precipitated cell mass solution, 24 well plates were dropped, 30. Mu.l per well, solidified at 37℃for 30min to gel, and cultured in colorectal cancer medium to obtain colorectal cancer organoids). As shown in fig. 8, compared with the control group, the colorectal cancer organ cultured by the self-made hydrogel of the present embodiment has a better state and shows a growing trend with the increase of time, which indicates that the hydrogel of the present embodiment meets the requirements of colorectal cancer organ culture and has a good capability of three-dimensional cell organ culture.
Example 4
The embodiment provides a preparation method of hydrogel and a culture method for culturing lung cancer organoids by using the hydrogel, which comprises the following steps:
S1, dissolving GelMA with the substitution degree of 30% in water, adding an initiator phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite (LAP), and uniformly mixing in a dark place to obtain a GelMA mixed solution with the final concentration of 5% and the LAP concentration of 0.05%;
S2, adding the type I collagen into the GelMA mixed solution, adding an initiator of tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate and sodium persulfate, uniformly mixing in a dark place, and filtering through a 0.22 micrometer filter membrane to obtain the GelMA-collagen mixed solution. Wherein the concentration of collagen is 0.5%, the concentration of ruthenium (II) complex is 0.001mol/L, and the concentration of sodium persulfate is 0.01mol/L;
S3, selecting lung cancer organoids to be passaged, adding pre-cooled PBS into a 24-pore plate, adding 1mL of PBS into each pore, collecting in a centrifuge tube, refrigerating at 4 ℃ for 15min, centrifuging (1000 rpm,5 min) to remove supernatant to obtain organoid precipitates, adding Tryple E for enzymolysis for 1min, centrifuging (1000 rpm,5 min) to remove supernatant to obtain precipitates, adding PBS for centrifuging (1000 rpm,5 min) again to remove redundant enzyme solution to obtain precipitates, and re-suspending the precipitates to form a cell mass solution.
S4, uniformly mixing the sterile GelMA-collagen mixed solution and the precipitated cell mass solution in a ratio of 85:15 (v/v), dripping a 24-pore plate, 30 microliters per pore, and curing for 15min in blue light to form gel. Adding lung cancer culture medium to culture to form lung cancer organoid after passage.
Further, the lung cancer organoids obtained in this example 4 were compared with the lung cancer organoids formed by commercial Matrigel of the control group (commercial Matrigel was uniformly mixed with the precipitated cell mass solution, 24 well plates were dropped, 30. Mu.l per well, solidified at 37℃for 30min to gel, and cultured in mouse lung medium to obtain mouse lung organoids). As shown in fig. 9, compared with the control group, the lung cancer organoid cultured by the self-made hydrogel of this example has a better status and shows a growing trend with the increase of time, which indicates that the hydrogel of this example meets the requirement of lung cancer organoid culture and has a good capability of three-dimensional cell culture organoids.
Example 5
The embodiment provides a preparation method of hydrogel and a culture method for culturing liver organs of mice by using the hydrogel, which comprises the following steps:
S1, dissolving GelMA with the substitution degree of 30% in water, adding an initiator phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite (LAP), and uniformly mixing in a dark place to obtain a GelMA mixed solution with the final concentration of 2% and the LAP concentration of 0.05%;
s2, adding the type I collagen into the GelMA mixed solution, adding an initiator of tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate and sodium persulfate, uniformly mixing in a dark place, and filtering by a 0.22 micrometer filter membrane to obtain the sterile GelMA-collagen mixed solution. Wherein the collagen concentration is 0.3%, the concentration of the ruthenium (II) complex is 0.0005mol/L, and the concentration of the sodium persulfate is 0.005mol/L;
S3, selecting primary extracted mouse liver organs, adding pre-cooled PBS into a 24-hole plate, adding 1mL of PBS into each hole, collecting into a centrifuge tube, refrigerating at 4 ℃ for 15min, centrifuging (1000 rpm,5 min) to remove supernatant to obtain organoid sediment, adding Tryple E for enzymolysis for 1min, centrifuging (1000 rpm,5 min) to remove supernatant to obtain sediment, adding PBS for centrifuging (1000 rpm,5 min) again to remove redundant enzyme solution to obtain sediment, and re-suspending the sediment to obtain a cell mass solution.
S4, uniformly mixing the sterile GelMA-collagen mixed solution and the precipitated cell mass solution in a ratio of 85:15 (v/v), dripping a 24-pore plate, 30 microliters per pore, and curing for 15min in blue light to form gel.
Further, the liver organoids obtained in this example 5 were compared with the liver organoids formed by commercial Matrigel of the control group (commercial Matrigel was uniformly mixed with the precipitated cell mass solution, 24 well plates were dropped, 30. Mu.l per well, solidified at 37℃for 30min to gel, and cultured in mouse liver medium to obtain mouse liver organoids). As shown in fig. 10, compared with the control group, the mice cultured by the self-made hydrogel of the embodiment have better liver organoid state, and show a growing trend with the increase of time, which indicates that the interpenetrating network hydrogel meets the requirement of liver organoid culture and has the capability of three-dimensional cell culture organoids.
The invention provides hydrogel and a preparation method thereof, organoids and a culture method thereof, and a drug detection method, which have the following beneficial effects:
the preparation method is simple, raw materials are easy to obtain, the preparation cost is low, each component corresponds to a photoinitiator in the preparation process, the photocuring crosslinking effect is effectively improved, an interpenetrating network structure is formed, and the mechanical strength of the hydrogel is effectively improved;
secondly, the hydrogel has a porous structure, the pore canal of the hydrogel is compact and the pore canal is rich, the three-dimensional growth of supporting cells is more facilitated, and the fibrous structure presented by the edge of the pore canal effectively improves the viscoelasticity of the hydrogel, and is more beneficial to changing the form, the precipitation matrix and the cell migration by overcoming the mechanical limitation in the organoid culture process;
Thirdly, when the hydrogel is used for organoid culture, a reliable in-vitro model is formed, the complexity of a human body specific microenvironment, an extracellular matrix and the like can be simulated, and the screening of effective medicaments for tumors can be realized.
It is to be understood that the above embodiments are merely illustrative of the application of the principles of the present invention, but not in limitation thereof. Various modifications and improvements may be made by those skilled in the art without departing from the spirit and substance of the invention, and are also considered to be within the scope of the invention.
Claims (10)
1. A method for preparing a hydrogel, the method comprising:
the preparation method comprises the steps of mixing a GelMA solution with a neutral collagen solution to obtain the GelMA-collagen mixed solution, wherein the GelMA solution is dissolved in water, a LAP photoinitiator is added and uniformly mixed in a dark place to obtain the GelMA solution;
The GelMA-collagen mixed solution is a neutral solution, the GelMA-collagen mixed solution comprises GelMA and LAP photoinitiators, type I collagen, tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photoinitiators and sodium persulfate photoinitiators,
In the GelMA-collagen mixed solution, the concentration of the tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate ranges from 0.0005 to 0.005mol/L;
The concentration range of the sodium persulfate photoinitiator is 0.005-0.05mol/L;
The concentration range of the LAP photoinitiator is 0.05% -0.5%;
the concentration range of the GelMA is 0.5% -20%;
the concentration range of the type I collagen is 0.05-1%;
The GelMA-collagen mixed solution is subjected to photo-curing to form transparent hydrogel with a first layer of cross-linked network and a second layer of cross-linked network which are staggered and interpenetrating, wherein the first layer of cross-linked network is formed by GelMA through photo-curing under the action of LAP photo-initiator;
The second layer of crosslinking network is formed by photocuring type I collagen under the action of a tri (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photoinitiator and a sodium persulfate photoinitiator,
When the concentration of GelMA is 0.5% -10% and the concentration of type I collagen is 0.05% -0.5%, the mechanical strength of the formed hydrogel is matched with that of organs or tissues with lower Young's modulus;
When the concentration of GelMA is 10% -20% and the concentration of type I collagen is 0.5% -1%, the mechanical strength of the formed hydrogel is matched with that of an organ or tissue with high Young's modulus.
2. The method of claim 1, wherein the time period of the photo-curing is in the range of 0.5 to 5 minutes.
3. A hydrogel produced by the production method according to claim 1 or 2.
4. A hydrogel is characterized by comprising a first layer of crosslinked network and a second layer of crosslinked network which is interlaced and interpenetrating with the first layer of crosslinked network, and the hydrogel is transparent,
The first layer of crosslinked network is formed by GelMA through photo-curing under the action of LAP photoinitiator;
The second layer of crosslinking network is formed by photocuring type I collagen under the action of a tri (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photoinitiator and a sodium persulfate photoinitiator;
in GelMA and LAP photoinitiator, a GelMA-collagen mixed solution formed by type I collagen, tris (2, 2 '-bipyridine) ruthenium (II) chloride hexahydrate photoinitiator and sodium persulfate photoinitiator, wherein the concentration of the tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate ranges from 0.0005 mol/L to 0.005mol/L;
The concentration range of the sodium persulfate photoinitiator is 0.005-0.05mol/L;
The concentration range of the LAP photoinitiator is 0.05% -0.5%;
the concentration range of the GelMA is 0.5% -20%;
the concentration range of the type I collagen is 0.05-1 percent, wherein,
When the concentration of GelMA is 0.5% -10% and the concentration of type I collagen is 0.05% -0.5%, the mechanical strength of the hydrogel is matched with organs or tissues with lower Young's modulus;
when the concentration of GelMA is 10% -20% and the concentration of type I collagen is 0.5% -1%, the mechanical strength of the hydrogel is matched with that of an organ or tissue with high Young's modulus.
5. A method of organoid culture, said method comprising:
The method for forming the cell mass solution of the organoid to be cultured comprises the steps of mixing GelMA solution with neutral collagen solution, wherein GelMA is dissolved in water, LAP photoinitiator is added, and the mixture is uniformly mixed in a dark place to obtain GelMA solution; uniformly mixing type I collagen with tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photoinitiator and sodium persulfate photoinitiator in a dark place to obtain a GelMA-collagen mixed solution, wherein cell clusters in the cell cluster solution are obtained by digestion and separation of tumor tissues or non-tumor tissues or by digestion and separation of tumor organoids or non-tumor organoids to be passaged;
the preparation method comprises the steps of obtaining a GelMA-collagen mixed solution, wherein the GelMA-collagen mixed solution is a neutral solution, the GelMA-collagen mixed solution comprises GelMA and LAP photoinitiator, type I collagen, tris (2, 2 '-bipyridine) ruthenium (II) hexahydrate photoinitiator and sodium persulfate photoinitiator, the GelMA-collagen mixed solution is a neutral solution, the substitution degree of GelMA in the GelMA-collagen mixed solution is 30% -95%, the concentration range of GelMA is 0.5% -20%, the concentration range of LAP photoinitiator is 0.05% -0.5%, the concentration range of type I collagen in the GelMA-collagen mixed solution is 0.05% -1%, the concentration range of tris (2, 2' -bipyridine) ruthenium (II) hexahydrate is 0.0005-0.005mol/L, and the concentration range of sodium persulfate photoinitiator is 0.005-0.05mol/L;
Mixing the GelMA-collagen mixed solution with the cell mass solution, adding a proper amount of mixed solution into a culture cavity or a culture hole for culturing the organoid, and performing photo-curing to form transparent organoid gel with a first layer of cross-linked network and a second layer of cross-linked network which are staggered and interpenetrating, wherein the first layer of cross-linked network is formed by photo-curing of the GelMA under the action of the LAP photo-initiator, and the second layer of cross-linked network is formed by photo-curing of the type I collagen under the action of the tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photo-initiator and the sodium persulfate photo-initiator;
Adding culture medium into culture cavity or culture hole containing the organoid gel, culturing to obtain tumor organoid or non-tumor organoid,
When the concentration of GelMA is 0.5% -10% and the concentration of type I collagen is 0.05% -0.5%, the mechanical strength of the organoid gel is matched with that of a tumor organoid or a non-tumor organoid with lower Young's modulus;
When the concentration of GelMA is 10% -20% and the concentration of type I collagen is 0.5% -1%, the mechanical strength of the organoid gel is matched with that of a tumor organoid or a non-tumor organoid with high Young's modulus.
6. The culture method according to claim 5, wherein the culture medium is selected from the group consisting of,
When the organoid is any one of liver, colorectal, pancreatic and pancreatic cancers, the concentration range of GelMA in the GelMA-collagen mixed solution is 0.5% -10%, and the concentration range of type I collagen is 0.05% -0.5%.
7. The culture method according to claim 5, wherein the culture medium is selected from the group consisting of,
When the organoid is any one of liver cancer and colorectal cancer, the concentration range of GelMA in the GelMA-collagen mixed solution is 10% -20%, and the concentration range of type I collagen is 0.5% -1%.
8. A method of culturing a lung or lung cancer organoid, said method comprising:
forming a lung or lung cancer cell mass solution, wherein the cell mass in the lung or lung cancer cell mass solution is obtained by digestion and separation of lung or lung cancer tissues or by digestion and separation of lung or lung cancer organoids to be passaged;
The preparation method comprises the steps of mixing a GelMA solution with a neutral collagen solution to obtain the GelMA-collagen mixed solution, wherein the GelMA solution is dissolved in water, LAP photo-initiator is added and uniformly mixed in a dark place to obtain the GelMA solution, the GelMA-collagen mixed solution is obtained by uniformly mixing I-type collagen with tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photo-initiator and sodium persulfate photo-initiator in a dark place, the GelMA-collagen mixed solution comprises GelMA, LAP photo-initiator, I-type collagen, tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photo-initiator and sodium persulfate photo-initiator, the GelMA-collagen mixed solution is a neutral solution, the substitution degree of GelMA in the GelMA-collagen mixed solution is 30% -95%, the concentration range of GelMA is 0.5% -10%, the concentration range of LAP photo-initiator is 0.05% -0.5%, the concentration range of I-type collagen in the GelMA-collagen mixed solution is 0.05% -0.05%, and the concentration range of tris (2, 2' -bipyridine) ruthenium (II) chloride photo-initiator is 0.005% -0.005 mol/sodium persulfate photo-initiator;
mixing the GelMA-collagen mixed solution with a lung or lung cancer cell mass solution, adding a proper amount of mixed solution into a culture cavity or a culture hole, and performing photo-curing to form transparent lung or lung cancer organoid gel with a first layer of cross-linked network and a second layer of cross-linked network which are staggered and interpenetrating, wherein the first layer of cross-linked network is formed by photo-curing the GelMA under the action of the LAP photo-initiator, and the second layer of cross-linked network is formed by photo-curing the type I collagen under the action of the tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photo-initiator and the sodium persulfate photo-initiator;
And adding a lung or lung cancer organoid culture medium into a culture cavity or a culture hole containing the lung or lung cancer organoid gel, and culturing to obtain the lung or lung cancer organoid.
9. A organoid, characterized in that it is cultivated by the cultivation method according to any one of claims 5 to 8.
10. A method of drug detection using the organoid of claim 9, said method of drug detection comprising:
introducing a drug to be tested into the organoid;
and obtaining the action result of the drug to be tested on the organoid.
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