CN117327300A - Hydrogel and preparation method thereof, organoid and culture method thereof, and drug detection method - Google Patents

Abstract

The invention proposes a hydrogel and its preparation method, organoids and its culture method, and drug detection method, which belong to the field of biomedicine 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' chloride) - Bipyridyl)ruthenium(II) hexahydrate photoinitiator and sodium persulfate photoinitiator; the GelMA-collagen mixed solution is photocured to form a first layer of cross-linked network and a second layer of cross-linked interpenetrating Network of hydrogels. 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 needs and monitoring of different types of organoids. Cell growth status requirements.

Description

Hydrogel and its preparation method, organoid and its culture method, drug detection method

Technical field

The invention belongs to the field of biomedicine technology, and specifically relates to a hydrogel and its preparation method, organoids and its culture method, and drug detection method.

Background technique

Organoids are self-organizing cell structures grown from pluripotent stem cells (PSCs) or pluripotent tissue-resident adult stem cells (ASCs). They are valuable models for studying organ development and human diseases, and provide useful tools for drug screening and toxicological studies. A variety of biomedical applications provide a valuable source of tissue.

Culture of organoids often relies on the use of tumor-derived basement membrane extract (BME). The BME hydrogel consists of a heterogeneous mixture of extracellular matrix (ECM) from mouse sarcoma, proteoglycans, and growth factors. However, BME hydrogels have weak mechanical properties and are difficult to adapt to various unique tumor microenvironments. Moreover, the heterogeneity of BME components limits the precise control of its physical and biochemical properties. The heterogeneous components and differences between batches greatly limit Clinical applications of cells grown in this matrix have been demonstrated, such as regenerative medicine and high-content screening.

Matrigel is currently used in organoid culture and can better meet the growth needs of various types of organoids. However, due to the unclear composition of Matrigel and the instability between batches, it greatly limits the large-scale and standardized organoid culture and organoid production. Use in organ drug susceptibility screening.

Synthetic hydrogels are used as matrix materials to support tissue regeneration, as cell or drug delivery carriers, or for cell culture. Most of the synthetic hydrogels currently proposed are single-component hydrogels, such as pure collagen, pure gelatin, Polyisonitrile polypeptide (PIC) and its modified products hydrogel or polyethylene glycol (PEG) hydrogel have low mechanical strength, insufficient cell growth sites, poor biocompatibility, and difficulty in meeting the convenience requirements. Issues such as the need to monitor cell growth status. At present, synthetic hydrogels composed of multiple components have also been proposed. However, they have problems such as relatively complex component preparation processes, poor biocompatibility, and single mechanical strength. When organoids are cultured, they have higher requirements on the mechanical properties and biocompatibility of the culture carrier, which poses a challenge to the use of synthetic hydrogels to simulate tissue-like three-dimensional structures and build microenvironments.

Contents of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art and provide a hydrogel and its preparation method, organoids and its culture method, and drug detection method.

In one aspect of the present invention, a preparation method of hydrogel is proposed, which preparation method includes:

Obtain 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; among which,

In the GelMA-collagen mixed solution, the concentration range of the tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate is 0.0005-0.005 mol/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% to 0.5%;

The concentration range of the GelMA is 0.5% to 20%;

The concentration range of type I collagen is 0.05-1%;

The GelMA-collagen mixed solution is photocured to form a hydrogel with an interpenetrating first layer of cross-linked network and a second layer of cross-linked network; wherein the first layer of cross-linked network is photoinitiated by GelMA in LAP Formed by light curing under the action of agent;

The second layer of cross-linked network is formed by photocuring type I collagen under the action of tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator and sodium persulfate photoinitiator.

Optionally, the photocuring time ranges from 0.5 to 5 minutes.

Another aspect of the present invention provides a hydrogel, which is prepared by the preparation method described above.

In another aspect of the present invention, a hydrogel is proposed, including a first layer of cross-linked network, and a second layer of cross-linked network interleaved and interpenetrating with the first layer of cross-linked network; wherein,

The first layer of cross-linked network is formed by photocuring GelMA under the action of LAP photoinitiator;

The second layer of cross-linked network is formed by light curing of type I collagen under the action of tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator and sodium persulfate photoinitiator;

In the GelMA-collagen mixed solution formed by GelMA, LAP photoinitiator, type I collagen, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator and sodium persulfate photoinitiator, The concentration range of the tris(2,2'-bipyridyl)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% to 0.5%;

The concentration range of the GelMA is 0.5% to 20%;

The concentration range of type I collagen is 0.05-1%.

In another aspect of the present invention, an organoid culture method is proposed, which culture method includes:

Forming a cell mass solution of organoids to be cultured; the cell mass in the cell mass solution is obtained by digestion and separation of tumor tissue or non-tumor tissue, or is obtained by digestion and separation of tumor organoids or non-tumor organoids to be passaged;

A GelMA-collagen mixed solution is obtained, 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 chloride) Ruthenium (II) hexahydrate photoinitiator and sodium persulfate photoinitiator; the GelMA-collagen mixed solution is a neutral solution; the GelMA substitution degree in the GelMA-collagen mixed solution is 30%-95 %, the GelMA concentration range is 0.5% to 20%, the LAP photoinitiator concentration range is 0.05% to 0.5%; the type I collagen concentration range in the GelMA-collagen mixed solution is 0.05 to 1 %, the concentration range of the tris(2,2'-bipyridyl)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 GelMA-collagen mixed solution is mixed with the cell mass solution, and an appropriate amount of the mixed solution is added to the culture chamber or culture hole used to culture the organoids, and is cured by light to form a first layer of interpenetrating interpenetrating layers. An organoid gel with a cross-linked network and a second layer of cross-linked network; wherein the first layer of cross-linked network is formed by light curing of the GelMA under the action of the LAP photoinitiator, and the second layer of cross-linked network is The network is formed by light curing of the type I collagen under the action of the tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator and the sodium persulfate photoinitiator;

Add culture medium to the culture chamber or culture well containing the organoid gel, and then culture it to obtain tumor organoids or non-tumor organoids.

Optionally, when the organoid is any one of liver, colorectum, pancreas, and pancreatic cancer, the GelMA concentration range in the GelMA-collagen mixed solution is 0.5% to 10%, and the type I collagen concentration range is 0.05～0.5%.

Optionally, when the organoid is either liver cancer or colorectal cancer, the GelMA concentration range in the GelMA-collagen mixed solution is 10% to 20%, and the concentration range of type I collagen is 0.5% to 1%. .

In another aspect of the present invention, a method for culturing lung or lung cancer organoids is proposed. The culturing method includes:

Forming a lung or lung cancer cell mass solution; the cell mass in the lung or lung cancer cell mass solution is obtained by digestion and separation of lung or lung cancer tissue, or by digestion and separation of lung or lung cancer organoids to be passaged;

Obtain GelMA-collagen mixed solution, which contains GelMA, LAP photoinitiator, type I collagen, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator and Sodium sulfate photoinitiator; the GelMA-collagen mixed solution is a neutral solution; the GelMA substitution degree in the GelMA-collagen mixed solution is 30%-95%, and the GelMA concentration range 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 trichloride (2,2' The concentration range of -bipyridyl)ruthenium(II) hexahydrate is 0.0005-0.005mol/L; the concentration range of the sodium persulfate photoinitiator is 0.005-0.05mol/L;

Mix the GelMA-collagen mixed solution with the lung or lung cancer cell mass solution, add an appropriate amount of the mixed solution into the culture chamber or culture hole, and undergo light curing to form a first layer of cross-linked network and a second layer of interpenetrating cross-linked network. A cross-linked network of lung or lung cancer organoid gel; wherein the first layer of cross-linked network is formed by light curing of the GelMA under the action of the LAP photoinitiator, and the second layer of cross-linked network is formed by the Type I collagen is formed by light curing under the action of the tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator and the sodium persulfate photoinitiator;

Add lung or lung cancer organoid culture medium to the culture chamber or culture hole containing the lung or lung cancer organoid gel, and then obtain lung or lung cancer organoid after culture.

Another aspect of the present invention provides an organoid, which is cultured and formed using the culture method described above.

In another aspect of the present invention, a method for detecting drugs using the organoids described above is proposed. The method for detecting drugs includes:

introducing the drug to be tested into the organoids;

Obtain the effect result of the drug to be tested on the organoid.

The invention proposes a hydrogel and its preparation method, organoids and its culture method, and drug detection method. The preparation method includes: obtaining a GelMA-collagen mixed solution; the GelMA-collagen mixed solution is a neutral solution, 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 light-cured to form an interpenetrating network hydrogel. 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 needs and requirements of different types of organoid tissues. Monitor cell growth status needs.

Description of drawings

Figure 1 is a flow chart of a hydrogel preparation method according to an embodiment of the present invention;

Figure 2 is a schematic structural diagram of a hydrogel according to another embodiment of the present invention;

Figure 3 is a flow chart of an organoid culture method according to another embodiment of the present invention;

Figure 4 is a flow chart of a lung or lung cancer organoid culture method according to another embodiment of the present invention;

Figure 5 is a flow chart of a method for detecting drugs using organoids according to another embodiment of the present invention;

Figure 6 is a scanning electron microscope observation picture of the self-made hydrogel in Example 1 of the present invention and the control group Matrigel; wherein, A and B in Figure 6 are the electron microscope pictures of the control group Matrigel at 1000x and 5000x respectively; Figure C and D in 6 are the electron microscope images of the self-made hydrogel in Example 1 at 1000x and 5000x respectively;

Figure 7 is a microscopic observation view of the use of self-made hydrogel and control group Matrigel in Example 2 of the present invention for culturing pancreatic cancer organoids;

Figure 8 is a microscopic observation of the use of homemade hydrogel and control group Matrigel in Example 3 of the present invention to culture colorectal cancer organoids;

Figure 9 is a microscopic observation of the use of homemade hydrogel and control group Matrigel in Example 4 of the present invention to culture mouse lung organoids;

Figure 10 is a microscopic observation of the use of homemade hydrogel and control group Matrigel in Example 5 of the present invention to culture mouse liver organoids.

Detailed ways

In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the described embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

As shown in Figure 1, one aspect of the present invention proposes a method S100 for preparing hydrogel for organoid culture, including steps S110 to S120:

S110. Obtain a GelMA-collagen mixed solution. The GelMA-collagen mixed solution is a neutral solution. The GelMA-collagen mixed solution contains GelMA, LAP photoinitiator, type I collagen, and tris(2,2'-bipyridyl)ruthenium chloride. (II) Hexahydrate photoinitiator and sodium persulfate photoinitiator.

It should be noted that in this embodiment, the photocuring agent corresponding to the GelMA component is LAP photoinitiator, and the photocuring agent corresponding to type I collagen is tris(2,2'-bipyridyl)ruthenium(II) chloride. For the hexahydrate photoinitiator and the sodium persulfate photoinitiator, the mixing order of each component is not specifically limited, as long as each component and the corresponding photoinitiator are mixed before photocuring. That is to say, the GelMA-collagen mixed solution includes a GelMA solution and a neutral collagen solution. GelMA and LAP photoinitiator can be mixed first to form a GelMA solution, and then type I collagen, tris(2,2'-bipyridyl chloride) Ruthenium (II) hexahydrate photoinitiator and sodium persulfate photoinitiator are mixed to form a neutral collagen solution, and then the two are mixed to form a GelMA-collagen mixed solution; the neutral collagen solution can also be formed first, and then the GelMA solution is formed, and then Mix the two to form a GelMA-collagen mixed solution. Of course, in addition to the above sequence, the two solutions can also be formed at the same time, or the formation process of one solution is synchronized with the mixing process of the two solutions. For example, type I collagen solution, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator, and sodium persulfate photoinitiator are added to the GelMA solution to obtain a GelMA-collagen mixed solution; Alternatively, the GelMA solution and LAP photoinitiator are added to the neutral collagen solution to obtain a GelMA-collagen mixed solution.

Exemplarily, in some preferred embodiments, the GelMA-collagen mixed solution formation process includes the following specific steps: dissolve GelMA in water, add phenyl-2,4,6-trimethylbenzoyl lithium phosphite ( LAP) photoinitiator, mix evenly in the dark to obtain a GelMA solution, add type I collagen to the GelMA solution, add tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator and persulfate Sodium photoinitiator, protect from light and mix evenly to obtain a GelMA-collagen mixed solution.

Illustratively, in other preferred embodiments, the GelMA-collagen mixed solution formation process includes the following specific steps: first dissolve type I collagen in an acetic acid solution, then add NaOH to adjust the pH to neutral, add initiator chlorination Tris(2,2'-bipyridyl)ruthenium(II) hexahydrate photoinitiator and sodium persulfate photoinitiator were mixed evenly in the dark to obtain a neutral collagen solution; then add GelMA to the neutral collagen solution and add A certain amount of LAP photoinitiator is mixed evenly in the dark to obtain a GelMA-collagen mixed solution.

In some preferred embodiments, type I collagen is naturally extracted animal collagen or recombinant collagen prepared by microbial methods.

In the GelMA-collagen mixed solution, the GelMA substitution degree is 30% to 95%; the GelMA concentration is 0.5% to 20%; the LAP photoinitiator concentration is 0.05% to 0.5%; the type I collagen concentration is 0.05 to 1%; The concentration of tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator is 0.0005~0.005mol/L; the concentration of sodium persulfate photoinitiator is 0.005~0.05mol/L. Wherein, the units of the above concentration ranges of LAP, GelMA and type I collagen are mass to volume ratio.

In some preferred embodiments, the concentration of GelMA may preferably be 0.5% to 10%, and the concentration of type I collagen may be preferably 0.05% to 0.5%. The mechanical strength of the hydrogel formed within the above preferred concentration range is higher than the Young's modulus. Matches organs or tissues with low Young's modulus, such as liver, colorectum, pancreatic cancer, and pancreatic cancer with Young's modulus of several kPa.

In other preferred embodiments, the concentration of GelMA may be preferably 10% to 20%, and the concentration of type I collagen may be preferably 0.5% to 1%. The mechanical strength and Young's modulus of the hydrogel formed within the above preferred concentration range are Matching with higher organs or tissues, for example, liver cancer, colorectal cancer, etc. whose Young's modulus is tens of kilopascals.

The S120, GelMA-collagen mixed solution is light-cured for 0.5 to 5 minutes to form a hydrogel with an interpenetrating network.

Specifically, in the GelMA-collagen mixed solution, the GelMA solution is light-cured to form a first layer of cross-linked network, the neutral collagen solution is light-cured to form a second layer of cross-linked network, and the first layer of cross-linked network is cross-linked with the second layer. Interconnected networks.

It should be noted that when using the above GelMA-collagen mixed solution for organoid culture, it is preferably filtered through a 0.22 micron filter membrane to obtain a sterile GelMA-collagen mixed solution, and then the mixed solution is mixed with the organoid cells to be subsequently cultured. The mass solution is mixed and photo-cross-linked to form a hydrogel with cells.

It should be further noted that when the hydrogel of this embodiment is used for organoid culture, the type I collagen in the hydrogel can be temperature-sensitively solidified as needed under organoid culture conditions to further increase cross-linking. degree, improves the mechanical strength of the hydrogel, eliminates the need to set up a separate heating step, greatly reduces the complexity and cost of the preparation process, and shortens the time of the preparation process.

The preparation process of the hydrogel of the present invention is simple. Based on the fact that each of the two components has its own photoinitiator, an interpenetrating network structure is formed through double cross-linking photo-curing polymerization, which effectively improves the degree of cross-linking and further effectively improves the hydrogel's properties. Mechanical strength; secondly, based on the adjustment of the concentration of each component during the preparation process, to form hydrogels with different mechanical strengths; thirdly, type I collagen in tris(2,2'-bipyridyl)ruthenium chloride ( II) Under the action of hexahydrate photoinitiator and sodium persulfate photoinitiator, it is light-cured to form a transparent hydrogel, which is convenient for observing the growth status of organoid tissue.

In another aspect of the present invention, a hydrogel for organoid culture is proposed. The hydrogel is prepared by the method described above. Please refer to the above description for the specific preparation process, which will not be described again here.

As shown in Figure 2, the hydrogel includes: a first layer of cross-linked network, and a second layer of cross-linked network that interpenetrates with the first layer of cross-linked network; among them, the first layer of cross-linked network is composed of GelMA under LAP light It is formed by photocuring under the action of initiator; the second layer of cross-linked network is composed of type I collagen in the presence of tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator and sodium persulfate photoinitiator. Formed by light curing.

In this embodiment, under light irradiation conditions, the GelMA double bond polymerization reaction forms the first layer of cross-linked network, and the tyrosine residues on type I collagen react by oxidative coupling reaction to form the second layer of cross-linked network. The two-layer network The structure is connected through the carboxyl groups on collagen and the amino groups on partially double-bonded GelMA, forming an interpenetrating network with strong mechanical properties.

It should be noted that during the preparation process of the hydrogel, GelMA, LAP photoinitiator, type I collagen, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator and sodium persulfate The photoinitiator is mixed to form a GelMA-collagen mixed solution, which is photocured to form a two-layer interpenetrating cross-linked network. In the GelMA-collagen mixed solution, the concentration range of tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate is 0.0005-0.005mol/L; the concentration range of sodium persulfate photoinitiator is 0.005 -0.05mol/L; the concentration range of LAP photoinitiator is 0.05%~0.5%; the concentration range of GelMA is 0.5%~20%; the concentration range of type I collagen is 0.05~1%.

In this embodiment, the hydrogel has an interpenetrating network structure and a porous structure, with dense pores and rich pore levels, which is conducive to supplementing the active sites required for specific cell growth and conducive to supporting the three-dimensional growth of cells; secondly, this embodiment The hydrogel has clear ingredients, including only GelMA and type I collagen. It is transparent and has the advantages of good biological activity, high mechanical strength and adjustability. It can meet the culture needs of different types of organoid tissues and overcome the existing problems of using collagen. It is difficult for hydrogels to simultaneously meet the requirements for high-definition observation and measurement of the growth status of organoid tissues and drug testing status, as well as the requirements for the growth activity of organoid tissues.

As shown in Figure 3, another aspect of the present invention proposes an organoid culture method S200, including the following steps S210 to S230:

S210. Form a cell mass solution of organoids to be cultured. The cell mass in the cell mass solution is obtained by digestion and isolation of primary tumor tissue or non-tumor tissue, or is digested from tumor organoids or non-tumor organoids to be passaged. Isolated.

It should be noted that in this embodiment, the organoids to be cultured may include non-tumor organoids, such as mouse pancreas, colorectum/small intestine, liver, etc., and may also be tumor organoids, such as liver cancer, colorectal cancer, etc. Cancer and other organoids. That is to say, digest pancreatic cancer tissue, colorectal cancer tissue, liver cancer tissue, etc. to obtain the corresponding cancer tissue, or digest mouse pancreas, colorectum/small intestine, liver, etc. to obtain normal tissue cell clusters, and then the corresponding cancer tissue The tissue or cell mass pellet is resuspended to obtain a cell mass solution.

Specifically, select the primary extraction or the above-mentioned organoids to be passaged, add pre-cooled PBS to the culture well plate, add 1 mL of PBS to each well, collect into a centrifuge tube, and refrigerate at 4°C for 15 min; centrifuge (1000 rpm, 5 min ), remove the supernatant, and obtain the organoid pellet; add Tryple E for enzymatic hydrolysis for 1 min; centrifuge (1000 rpm, 5 min), remove the supernatant, and obtain the pellet; add PBS and centrifuge again (1000 rpm, 5 min), remove excess enzyme solution, and obtain the pellet. The pellet was then resuspended to obtain a cell pellet solution.

S220. Obtain a GelMA-collagen mixed solution, which contains GelMA, LAP photoinitiator, type I collagen, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator, and Sodium persulfate photoinitiator, wherein the degree of substitution of GelMA in the GelMA-collagen mixed solution is 30%-95%, the concentration range of GelMA is 0.5%-20%, and 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%, and the concentration range of tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate is 0.0005-0.005mol/L; The concentration range of sodium sulfate photoinitiator is 0.005-0.05mol/L. Please refer to the previous record for the specific formation process.

S230. Mix the GelMA-collagen mixed solution and the cell mass solution, add an appropriate amount of the mixed solution into the culture chamber or culture well for culturing organoids, and form an organoid gel through light curing.

Specifically, the GelMA-collagen mixed solution and the cell cluster precipitation solution were mixed at a ratio of 85:15 (v/v) to form a cell-gel mixed solution, and the cell-gel mixed solution was mixed with 5 to 30 microns per well. Liter and 50 to 500 organoids per well are added to the culture wells or chambers, and then irradiated under blue light for 0.5-5 minutes to cross-link into a gel to form an organoid gel containing cells.

Among them, in the process of cross-linking to form a gel, in the mixture of GelMA-collagen mixed solution and cell mass solution, GelMA is photo-cured under the action of LAP photoinitiator to form the first layer of cross-linked network, and type I collagen is cured in trichloride. (2,2'-bipyridyl)ruthenium(II) hexahydrate photoinitiator and sodium persulfate photoinitiator are photocured to form a second layer of cross-linked network. The first cross-linked network and the second cross-linked network are The network interpenetrates, i.e., forms an organoid gel containing interpenetrating networks of cells.

S240. Add culture medium to the culture chamber or culture well containing the organoid gel, and culture to obtain tumor organoids or non-tumor organoids.

Specifically, the corresponding organoid culture medium is added to the above-mentioned culture well or culture chamber, placed in an incubator at 37° C., 5% CO ₂ , and 95% humidity for culture to perform organoid expansion culture.

Further, digest the amplified organoids to obtain organoid cell clusters, resuspend them in GelMA-collagen mixed solution, add 10 to 50 organoids per well into the culture chamber or culture well, and irradiate with blue light for 0.5-5 minutes. Glue, add the corresponding organoid culture medium, place it in an incubator at 37°C, 5% CO ₂ and 95% humidity for culture. After 1 to 3 days, replace the culture medium containing the drug to be tested, and conduct cell activity after 5 to 10 days of culture. detection.

It should be noted that when culturing organoids at the above culture temperature, the type I collagen in the hydrogel can be further temperature-sensitive cross-linked as needed to improve the mechanical strength of the hydrogel.

It should be further noted that in this embodiment, for organoids with different Young's modulus, the concentration of each component can be adjusted during the hydrogel preparation process to form hydrogels with different mechanical strengths to match different Young's modulus of organoids.

For example, when the organoids are those with a low Young's modulus (several kPa), such as liver, colorectum, pancreas/pancreatic cancer, etc., the preparation method of the organoid gel is as follows:

S1. Dissolve GelMA in water, add the initiator phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP), and mix evenly in the dark to obtain a GelMA mixed solution; add type I collagen to the GelMA and mix In the solution, add the initiator tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfate, mix evenly in the dark, and pass through a 0.22 micron filter membrane to obtain a sterile GelMA-collagen mixed solution;

In step S1, in the GelMA mixed solution, the GelMA substitution degree is 30% to 95%, the GelMA concentration is 0.5% to 10%, the concentration of the initiator LAP is 0.05% to 0.5%, in the GelMA-collagen mixed solution, The concentration of collagen is 0.05-0.5%, the concentration of initiator tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate is 0.0005-0.005mol/L, and the concentration of sodium persulfate is 0.005-0.05mol/ L.

S2. Select primary extraction or organoids to be passaged, add pre-cooled PBS to the culture well plate, add 1mL PBS to each well, collect into a centrifuge tube, refrigerate at 4°C for 15 minutes; centrifuge (1000rpm, 5min), Remove the supernatant to obtain the organoid pellet; add Tryple E for enzymatic digestion for 1 minute; centrifuge (1000 rpm, 5 min) to remove the supernatant and obtain the precipitate; add PBS and centrifuge again (1000 rpm, 5 min) to remove excess enzyme solution and obtain the precipitate. Suspend to obtain cell mass solution;

S3. Mix the sterile GelMA-collagen mixed solution and the cell mass solution at a ratio of 85:15 (v/v), and drip the gel onto the culture well plate, 5 to 30 microliters per well, to obtain a cell-gel mixed solution. ; Cross-link under blue light irradiation for 0.5 to 5 minutes to obtain organoid gel, add corresponding culture medium for culture and observation.

For example, when the organoids are those with a high Young's modulus (tens of kilopascals), such as liver cancer, colorectal cancer, etc., the preparation method of the organoid gel is as follows:

S1. Dissolve GelMA in water, add the initiator phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP), and mix evenly in the dark to obtain a GelMA mixed solution; add type I collagen to the GelMA and mix In the solution, add the initiator tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfate, mix evenly in the dark, and pass through a 0.22 micron filter membrane to obtain a sterile GelMA-collagen mixed solution;

In step S1, in the GelMA mixed solution, the GelMA substitution degree is 30% to 95%, the GelMA concentration is 10% to 20%, the concentration of the initiator LAP is 0.05% to 0.5%, in the GelMA-collagen mixed solution, The concentration of collagen is 0.5-1%, the concentration of initiator tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate is 0.0005-0.005mol/L, and the concentration of sodium persulfate is 0.005-0.05mol/ L.

S2. Select primary extraction or organoids to be passaged, add pre-cooled PBS to the culture well plate, add 1mL PBS to each well, collect into a centrifuge tube, refrigerate at 4°C for 15 minutes; centrifuge (1000rpm, 5min), Remove the supernatant to obtain the organoid pellet; add Tryple E for enzymatic digestion for 1 minute; centrifuge (1000 rpm, 5 min) to remove the supernatant and obtain the precipitate; add PBS and centrifuge again (1000 rpm, 5 min) to remove excess enzyme solution and obtain the precipitate. Suspend to obtain cell mass solution;

S3. Mix the sterile GelMA-collagen mixed solution and the cell mass solution at a ratio of 85:15 (v/v), and drip the gel onto the culture well plate, 5 to 30 microliters per well, to obtain a cell-gel mixed solution. ; Cross-link under blue light irradiation for 0.5 to 5 minutes to obtain organoid gel, add corresponding culture medium for culture and observation.

In this embodiment, when interpenetrating network hydrogel is used to culture organoids, a reliable in vitro model of organoids is formed. The culture method is simple and easy to operate. By adjusting the concentration of the components in the hydrogel, it can form a reliable organoid model with different mechanical properties. Strong hydrogel to meet the mechanical strength and biological activity required by different organoids.

As shown in Figure 4, another aspect of the present invention proposes a lung or lung cancer organoid culture method S300, including the following steps S310 to S340:

S310. Form 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 tissue, or by digestion and separation of lung or lung cancer organoids to be passaged;

S320. Obtain a GelMA-collagen mixed solution, which contains GelMA, LAP photoinitiator, type I collagen, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator, and Sodium persulfate photoinitiator; GelMA-collagen mixed solution is a neutral solution; among them, the substitution degree of GelMA in the GelMA-collagen mixed solution is 30%-95%, and the GelMA concentration range is 0.5%-20%. LAP photoinitiation The concentration range of the agent is 0.05% ~ 0.5%; the concentration range of type I collagen in the GelMA-collagen mixed solution is 0.05 ~ 1%, and the concentration range of tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate The range is 0.0005-0.005mol/L; the concentration range of sodium persulfate photoinitiator is 0.005-0.05mol/L.

S330. Mix the GelMA-collagen mixed solution with the lung or lung cancer cell mass solution, add an appropriate amount of the mixed solution into the culture chamber or culture well, and undergo light curing to form a lung or lung cancer organoid gel.

S340. Add the lung or lung cancer organoid culture medium to the culture chamber or culture well containing the lung or lung cancer organoid gel, and after culturing, the lung or lung cancer organoid is obtained.

It should be noted that the culture process of lung or lung cancer organoids in this embodiment is the same as the culture process of other organoids described above. Please refer to the above description and will not be repeated here.

In another aspect of the present invention, an organoid is proposed. A variety of organoids can be cultured using the organoid culture method described above. Please refer to the above description for the specific culture method, which will not be described again here.

Exemplarily, organoids include non-tumor organoids and tumor organoids, wherein non-tumor organoids include pancreatic organoids, colorectal organoids, small intestinal organoids, lung organoids, and liver organoids; tumor organoids include There are pancreatic cancer organoids, colorectal cancer organoids, lung cancer organoids, liver cancer organoids.

In some preferred embodiments, the organoids include pancreatic cancer organoids, rectal cancer organoids, lung cancer organoids, lung organoids, liver organoids, etc. Of course, other organoids can also be cultured according to actual needs, which are not mentioned here. List them one by one.

As shown in Figure 5, another aspect of the present invention proposes a method S400 for drug detection using organoids, including steps S410 to S420:

S410. Introduce the drug to be tested into the organoids;

S420. Obtain the effect results of the drug to be tested on the organoids.

In this embodiment, based on the above-mentioned organoid in vitro model, the complexity of the human body's specific microenvironment, extracellular matrix, etc. can be simulated to realize drug screening, especially effective drugs for pancreatic cancer, rectal cancer, lung cancer and other cancers.

Exemplarily, conventional drug sensitivity testing for tumor organoids on well plates includes the following steps:

(1) Prepare a drug-containing culture medium of appropriate concentration,

(2) Tumor cancer organoids are placed in culture wells in a non-dynamic culture environment, such as the culture wells of a 96-well plate;

(4) Observe and record the growth status of the tumor organoids, aspirate the culture medium in the culture wells, add 100 μL of culture medium containing the drug solution to each well, and place it in an incubator at 37°C and 5% CO2 for culture;

(4) Observe and record the growth status of tumor organoids on days 3-5;

(5) Use CTG detection kit for activity detection. Add 100 μL CTG detection reagent to each well, shake with a shaker for 5 minutes, and incubate at room temperature for 25 minutes; after the incubation, use a chemiluminescence microplate reader for detection.

Exemplarily, routine drug sensitivity testing on tumor organoids under dynamic culture includes the following steps:

(1) Prepare a drug-containing culture medium of appropriate concentration:

(2) The cultured tumor organoids are placed in a culture chamber in a dynamic culture environment. For example, a chip with three connected chambers is used, and the tumor organoids are placed in the middle chamber;

(3) Observe and record the growth status of tumor organoids. Aspirate the culture medium in the chip and add the culture medium containing the drug solution. Add 50 μL culture medium to the wells on the left and right sides of the chip, and add 30 μL culture medium to the middle well;

(4) After adding the drug, put the chip into the swing perfusion instrument at 3°/120min for flow culture;

(5) Observe and record the growth status of tumor organoids from days 3 to 5, and add 30 μL of drug-containing culture medium to the middle well;

(6) Use CTG detection kit for activity detection. Discard all the culture medium from both sides of the wells, add 20 μL CTG dilution (1:1 dilution of CTG reagent stock solution and culture medium), add 50 μL CTG stock solution, mix by pipetting, shake with a shaker for 5 minutes, and incubate at room temperature for 25 minutes. Take out the lysate and place it into a 96-well plate with completely opaque sides and bottom. Detection was performed using a chemiluminescence microplate reader.

The preparation method of hydrogel and the culture method of organoids will be further described below with reference to several specific examples:

Example 1

This example provides a method for preparing GelMA-collagen interpenetrating network hydrogel, including:

S1. Dissolve GelMA with a substitution degree of 60% in water, add the initiator phenyl-2,4,6-trimethylbenzoyllithium phosphite (LAP), and mix evenly in the dark to obtain a final concentration of 2% GelMA mixed solution, LAP concentration 0.05%;

S2. Add type I collagen to the GelMA mixed solution, add the initiator tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfate, and mix evenly in the dark to obtain a GelMA-collagen mixed solution. , where the collagen concentration is 0.2%, the ruthenium (II) complex concentration is 0.0005mol/L, and the sodium persulfate concentration is 0.005mol/L;

S3. Cross-link the above GelMA-collagen mixed solution under blue light irradiation for 1 minute to obtain the GelMA-collagen interpenetrating network hydrogel, whose structure is shown in Figure 2.

Further, the hydrogel obtained in Example 1 was compared with the commercial Matrigel of the control group (the commercial Matrigel was prepared by curing at 37°C for 30 minutes). The hydrogel obtained in Example 1 was compared with the two hydrogels obtained in this Example. After the gel was freeze-dried, it was observed with a scanning electron microscope. The results are shown in Figure 6. Compared with the matrigel in the control group, in this example, the porous structure of the hydrogel has dense pores and rich pore levels, which is conducive to the three-dimensional growth of supporting cells, and the edge structure shape of the pores is The non-smooth fibrous shape effectively improves the viscoelasticity of the hydrogel, which is more conducive to cells overcoming mechanical constraints to change shape, precipitate matrix, and migrate during organoid culture.

Example 2

This example provides a method for preparing hydrogel and a method for using it to culture pancreatic cancer organoids, including:

S1. Dissolve GelMA with a substitution degree of 60% in water, add the initiator phenyl-2,4,6-trimethylbenzoyllithium phosphite (LAP), and mix evenly in the dark to obtain a final concentration of 10% GelMA mixed solution, LAP concentration 0.1%;

S2. Add type I collagen to the GelMA mixed solution, add the initiator tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfate, mix evenly in the dark, and filter through a 0.22 micron filter. Obtain sterile GelMA-collagen mixed solution. 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. Select the pancreatic cancer organoids to be passaged, add pre-cooled PBS to the 24-well plate, add 1 mL PBS to each well, collect into centrifuge tubes, refrigerate at 4°C for 15 minutes, then centrifuge (1000 rpm, 5 minutes), remove the supernatant The supernatant was added to obtain the organoid precipitate; Tryple E was added for enzymatic hydrolysis for 1 min, then centrifuged (1000 rpm, 5 min), the supernatant was removed, and the precipitate was obtained; PBS was added and centrifuged again (1000 rpm, 5 min) to remove excess enzyme solution and the precipitate was obtained, and Resuspend the pellet to form a cell pellet solution.

S4. Mix the sterile GelMA-collagen mixed solution and the precipitated cell cluster solution at a ratio of 85:15 (v/v), glue onto a 24-well plate, 30 microliters per well, and solidify under blue light for 15 minutes to form a gel.

Further, the pancreatic cancer organoids obtained in Example 2 and the pancreatic cancer organoids formed by commercial Matrigel in the control group (commercial Matrigel) were evenly mixed with the precipitated cell cluster solution, and the gel was dripped into a 24-well plate. Each well was 30 microliters, solidified at 37°C for 30 minutes to form a gel, added to pancreatic cancer culture medium for culture, and obtained pancreatic cancer organoids) for comparison. As shown in Figure 7, compared with the control group, the pancreatic cancer organoids cultured with the self-made hydrogel of this embodiment are in better condition and show an increasing trend with time, which shows that the hydrogel of this embodiment meets the requirements. The need for pancreatic cancer organoid culture and the ability to culture organoids from three-dimensional cells.

Example 3

This example provides a method for preparing hydrogel and a method for using it to culture colorectal cancer organoids, including:

S1. Dissolve GelMA with a substitution degree of 60% in water, add the initiator phenyl-2,4,6-trimethylbenzoyllithium phosphite (LAP), and mix evenly in the dark to obtain a final concentration of 12% GelMA mixed solution, LAP concentration 0.2%;

S2. Add type I collagen to the GelMA mixed solution, add the initiator tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfate, mix evenly in the dark, and filter through a 0.22 micron filter. Obtain sterile GelMA-collagen mixed solution. 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. Select the primary extracted colorectal cancer organoids, add pre-cooled PBS to the 24-well plate, add 1mL PBS to each well, collect into centrifuge tubes, refrigerate at 4°C for 15 minutes, and then centrifuge (1000rpm, 5min) Remove the supernatant to obtain the organoid precipitate; add Tryple E for enzymatic digestion for 1 minute, then centrifuge (1000 rpm, 5 min) to remove the supernatant and obtain the precipitate; add PBS and centrifuge again (1000 rpm, 5 min) to remove excess enzyme solution and obtain the precipitate. The pellet was then resuspended to form a cell pellet solution.

S4. Mix the sterile GelMA-collagen mixed solution and the precipitated cell cluster solution at a ratio of 85:15 (v/v), glue onto a 24-well plate, 30 microliters per well, and solidify under blue light for 15 minutes to form a gel.

Further, the colorectal cancer organoids obtained in Example 3 and the colorectal cancer organoids formed by the commercial Matrigel in the control group (commercial Matrigel and the precipitated cell mass solution were evenly mixed, and placed on a 24-well plate, 30 microliters per well, solidified at 37°C for 30 minutes to form a gel, added colorectal cancer culture medium for culture, and obtained colorectal cancer organoids) for comparison. As shown in Figure 8, compared with the control group, the colorectal cancer organs cultured by the self-made hydrogel of this embodiment are in better condition and show an increasing trend as time goes by, which shows that the hydrogel of this embodiment satisfies the requirements of rectal cancer. The need for cancer organoid culture and the ability to have good three-dimensional cell culture organoids.

Example 4

This example provides a method for preparing hydrogel and a method for cultivating lung cancer organoids, including:

S1. Dissolve GelMA with a substitution degree of 30% in water, add the initiator phenyl-2,4,6-trimethylbenzoyllithium phosphite (LAP), and mix evenly in the dark to obtain a final concentration of 5% GelMA mixed solution, LAP concentration 0.05%;

S2. Add type I collagen to the GelMA mixed solution, add the initiator tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfate, mix evenly in the dark, and filter through a 0.22 micron filter. A GelMA-collagen mixed solution was obtained. 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. Select lung cancer organoids to be passaged, add pre-cooled PBS to the 24-well plate, add 1 mL PBS to each well, collect into centrifuge tubes, refrigerate at 4°C for 15 minutes, and then centrifuge (1000 rpm, 5 minutes) to remove the supernatant. , to obtain organoid precipitates; add Tryple E for enzymatic hydrolysis for 1 minute, then centrifuge (1000 rpm, 5 min) to remove the supernatant, and obtain a precipitate; add PBS and centrifuge again (1000 rpm, 5 min) to remove excess enzyme solution and obtain a precipitate, and then recycle the precipitate. Resuspend to form a cell pellet solution.

S4. Mix the sterile GelMA-collagen mixed solution and the precipitated cell cluster solution at a ratio of 85:15 (v/v), glue onto a 24-well plate, 30 microliters per well, and solidify under blue light for 15 minutes to form a gel. Add lung cancer culture medium for culture to form lung cancer organoids after passage.

Further, the lung cancer organoids obtained in Example 4 and the lung cancer organoids formed by the commercial Matrigel in the control group (commercial Matrigel and the precipitated cell cluster solution were evenly mixed, and glued into a 24-well plate, 30 microns per well) liter, solidified at 37°C for 30 minutes to form a gel, and then added mouse lung culture medium for culture to obtain mouse lung organoids) for comparison. As shown in Figure 9, compared with the control group, the lung cancer organoids cultured by the self-made hydrogel of this embodiment are in better condition and show an increasing trend with time, which shows that the hydrogel of this embodiment meets the requirements of lung cancer organoids. The demand for organ culture has good ability to culture organoids from three-dimensional cells.

Example 5

This example provides a method for preparing hydrogel and a method for using it to culture mouse liver organoids, including:

S1. Dissolve GelMA with a substitution degree of 30% in water, add the initiator phenyl-2,4,6-trimethylbenzoyllithium phosphite (LAP), and mix evenly in the dark to obtain a final concentration of 2% GelMA mixed solution, LAP concentration 0.05%;

S2. Add type I collagen to the GelMA mixed solution, add the initiator tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfate, mix evenly in the dark, and filter through a 0.22 micron filter. Obtain sterile GelMA-collagen mixed solution. The concentration of collagen is 0.3%, the concentration of ruthenium (II) complex is 0.0005mol/L, and the concentration of sodium persulfate is 0.005mol/L;

S3. Select the primary extracted mouse liver organoids, add pre-cooled PBS to the 24-well plate, add 1mL PBS to each well, collect into a centrifuge tube, refrigerate at 4°C for 15 minutes, and then centrifuge (1000 rpm, 5 minutes) Remove the supernatant to obtain the organoid precipitate; add Tryple E for enzymatic digestion for 1 minute, then centrifuge (1000 rpm, 5 min) to remove the supernatant and obtain the precipitate; add PBS and centrifuge again (1000 rpm, 5 min) to remove excess enzyme solution and obtain the precipitate. The pellet was then resuspended to obtain a cell pellet solution.

S4. Mix the sterile GelMA-collagen mixed solution and the precipitated cell cluster solution at a ratio of 85:15 (v/v), and glue onto a 24-well plate, with 30 microliters per well, and solidify under blue light for 15 minutes to form a gel.

Further, the liver organoids obtained in Example 5 and the liver organoids formed by commercial Matrigel in the control group (commercial Matrigel) were evenly mixed with the precipitated cell cluster solution, and glued into a 24-well plate, with 30 microns per well. liter, solidified at 37°C for 30 minutes to form a gel, and then added mouse liver culture medium for culture to obtain mouse liver organoids) for comparison. As shown in Figure 10, compared with the control group, the mouse liver organoids cultured by the self-made hydrogel in this example were in better condition and showed an increasing trend with time, indicating that the interpenetrating network hydrogel meets the requirements of the liver organoids. The need for organoid culture and the ability to culture organoids from three-dimensional cells.

The present invention proposes a hydrogel and its preparation method, organoids and its culture method, and drug detection method, which have the following beneficial effects:

First, the preparation method of the present invention is simple, the raw materials are easy to obtain, and the preparation cost is low. During the preparation process, each component corresponds to a photoinitiator, which effectively improves the photocuring cross-linking effect and forms an interpenetrating network structure. Effectively improve the mechanical strength of hydrogel;

Second, the hydrogel of the present invention has clear ingredients and very good biocompatibility, and the hydrogel structure has a first layer of cross-linked network structure and a second layer of cross-linked network structure that are intertwined with each other, and It has a porous structure with dense pores and rich pore levels, which is conducive to supplementing the active sites required for specific cell growth and conducive to cell growth and proliferation. Secondly, the hydrogel has a porous structure with dense pores and rich pore levels, which is more conducive to supporting cells. Three-dimensional growth, and the fibrous structure present at the edge of the pore channel effectively improves the viscoelasticity of the hydrogel, which is more conducive to cells overcoming mechanical constraints to change shape, precipitate matrix, and cell migration during organoid culture;

Third, when the hydrogel of the present invention is used for organoid culture, it forms a reliable in vitro model, which can simulate the complexity of the human body's specific microenvironment, extracellular matrix, etc., and enable the screening of effective drugs for tumors.

It can be understood that the above embodiments are only exemplary embodiments adopted to illustrate the principles of the present invention, but the present invention is not limited thereto. For those of ordinary skill in the art, various modifications and improvements can be made without departing from the spirit and essence of the present invention, and these modifications and improvements are also regarded as the protection scope of the present invention.

Claims (10)

1. A method for preparing a hydrogel, the method comprising:

obtaining a GelMA-collagen mixed solution;

the GelMA-collagen mixed solution is a neutral solution, and comprises GelMA and LAP photoinitiators, type I collagen, tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photoinitiators and sodium persulfate photoinitiators; wherein,

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; the first layer of crosslinking network is formed by GelMA through photo-curing under the action of LAP photoinitiator;

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.

2. The method of claim 1, wherein the time for photocuring 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 comprising a first layer of crosslinked network and a second layer of crosslinked network interlaced interpenetrating with 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%.

5. A method of organoid culture, said method comprising:

forming a cell mass solution of the organoid to be cultured; 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 tumor organoids or non-tumor organoids to be passaged;

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; 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%, and 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) chloride 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 the cell mass solution, adding a proper amount of mixed solution into a culture cavity or a culture hole for culturing the organoids, 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; the first layer of crosslinking network is formed by photocuring of the GelMA under the action of the LAP photoinitiator, and the second layer of crosslinking network is formed by photocuring of the type I collagen under the action of the tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photoinitiator and the sodium persulfate photoinitiator;

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.

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 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%.

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; 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;

obtaining a GelMA-collagen mixed solution, wherein the GelMA-collagen mixed solution comprises GelMA and LAP photoinitiators, 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%, and 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%, and 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;

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; the first layer of crosslinking network is formed by photocuring of the GelMA under the action of the LAP photoinitiator, and the second layer of crosslinking network is formed by photocuring of the type I collagen under the action of the tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate photoinitiator and the sodium persulfate photoinitiator;

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.