CN114231490A

CN114231490A - Method for culturing tumor organoid by permeable hydrogel scaffold

Info

Publication number: CN114231490A
Application number: CN202111589299.5A
Authority: CN
Inventors: 张函槊; 刘勇
Original assignee: Genex Health Co Ltd
Current assignee: Genex Health Co Ltd
Priority date: 2021-12-23
Filing date: 2021-12-23
Publication date: 2022-03-25
Anticipated expiration: 2041-12-23
Also published as: CN114231490B

Abstract

The invention discloses a method for culturing tumor organoids by using a permeable hydrogel scaffold. The invention provides a method for culturing tumor organoids, which comprises the following steps: mixing tumor cells to be cultured with hydrogel matrix to make the final concentration of cells be (5-10) × 10³Per mL; adding coagulant and protein crosslinking agent, mixing to obtain hydrogel solution, adding into the bottom of cell filter with pore diameter of 10-70 μm, standing at 37 deg.C until hydrogel solidifies, and adding tumor organoid culture solution above the hydrogel; and horizontally placing the cell filter on a tube opening of a centrifuge tube which can be matched with the cell filter, and vertically placing and culturing the centrifuge tube to obtain the tumor organoid. The invention makes the culture solution continuously and slowly permeate downwards through the loose porous structure of the hydrogel under the action of gravity, the cells are fully supported by nutrition and take off metabolites in time, the culture success rate is improved, and the culture period of tumor organoids is shortened.

Description

Method for culturing tumor organoid by permeable hydrogel scaffold

Technical Field

The invention relates to the field of biomedicine, in particular to a method for culturing tumor organoids by using a permeable hydrogel scaffold.

Background

The tumor organoid is a miniature three-dimensional cell model which is cultured by taking tumor cells of tumor tissues of a patient and proper biological materials as three-dimensional culture matrixes and simulating the environment of the tumor cell matrixes. Tumor organoids have great potential in preclinical screening of anticancer drugs, drug response, and prognosis prediction of patients.

The hydrogel is a scaffold matrix commonly used for culturing tumor organoids, and is mainly derived from synthetic or natural materials such as alginate, gelatin, hyaluronic acid, agarose, laminin, collagen or fibrin. The commercialized Matrigel culture has the most clear effect, realizes successful culture of various tumor organoids, and is widely applied due to the limitation of high price. In the process of culturing tumor organoids, the hydrogel scaffold is prepared by mixing tumor cells and hydrogel matrix, then paving the mixture fully or dripping the mixture on the bottom of a cell culture plate hole, and adding corresponding organoid culture solution after hydrogel is solidified. The method for culturing organoids by loading hydrogel scaffolds on the bottoms of wells of cell culture plates has the following disadvantages: firstly, the culture solution is gradually diffused and infiltrated into the hydrogel only by virtue of the loose and porous structure of the hydrogel, so that the exchange efficiency of nutrient substances and metabolites is low, and the cells are slowly grown or stagnated; secondly, once the sponge-like structure with loose and porous hydrogel is saturated by the culture solution, external fresh culture solution can not permeate, so that cells die due to lack of nutrition and accumulation of local metabolites; finally, the expensive organoid culture fluid may not be fully utilized and may be discarded by the replacement fluid, resulting in waste.

Disclosure of Invention

In order to overcome the defects, the permeable hydrogel scaffold prepared by taking sheep plasma as a matrix material is used for culturing tumor organoids.

In a first aspect, the invention claims a method of culturing (in vitro culture) tumor organoids.

The method for culturing (in vitro culturing) tumor organoids claimed by the invention can comprise the following steps:

(A1) mixing tumor cells to be cultured with hydrogel matrix to give final cell concentration of 5-10 × 10³Per mL (e.g., 5X 10)³/mL)；

(A2) Adding a coagulant and a protein cross-linking agent into the (A1), and mixing to obtain a hydrogel solution containing tumor cells;

(A3) placing the hydrogel solution containing tumor cells at 20-25 deg.C until it becomes viscous, adding into the bottom of cell filter with pore diameter of 10-70 μm (such as 40 μm), standing at 37 deg.C until the hydrogel solution containing tumor cells solidifies, and adding tumor organoid culture solution above the hydrogel;

(A4) placing the cell filter (A3) containing the hydrogel and the culture solution horizontally on a tube opening of a centrifuge tube capable of being matched with the cell filter (clamping the cell filter at the tube opening, as shown in figure 1), and then placing the centrifuge tube vertically for culturing to obtain the tumor organoid.

In the step (a1), the hydrogel matrix may be selected from any one of the following: isolated sheep plasma or other mammalian or human plasma, alginate, gelatin, hyaluronic acid, agarose, laminin, collagen, fibrin, Matrigel, and other synthetic or natural materials.

In a particular embodiment of the invention, the hydrogel matrix is ex vivo sheep plasma.

In the step (a2), the coagulant is any one or more of inorganic salt or/and organic salt containing calcium ions;

further, the coagulant is selected from any one or more of: calcium chloride, calcium gluconate, calcium dihydrogen phosphate, calcium nitrate, calcium bicarbonate, calcium hydrogen sulfate, calcium hydrogen sulfite and calcium hypochlorite.

In a particular embodiment of the invention, the accelerator is calcium chloride. The concentration of the calcium chloride in the hydrogel solution is 3-6mmol/L (e.g., 4.5 mmol/L).

In step (a2), the protein cross-linking agent may be selected from any one or more of: glutaraldehyde, EDC, SMCC, DSS, DST, MBS, SPDP.

In a particular embodiment of the invention, the protein cross-linking agent is glutaraldehyde. The glutaraldehyde is present in the hydrogel solution in an amount of 0.1 to 0.5 percent by volume (e.g., 0.25 percent).

In step (a2), the calcium chloride and the glutaraldehyde are added in the form of a 100 × stock solution.

In step (A3), the tumor cell-containing hydrogel solution is left at room temperature for 3-5min (e.g., 3min) and then added to the bottom of a cell filter having a pore size of 10-70 μm (e.g., 40 μm).

In step (A3), the hydrogel solution containing tumor cells is added to the bottom of the cell filter, and then kept at 37 ℃ for 5-7min (e.g., 7 min).

In step (a3), the volume of the tumor organoid culture solution added above the hydrogel is equal to the volume of the hydrogel (or the hydrogel solution).

In the step (A4), the culturing is carried out at 37 ℃ with 5% CO₂Culturing under the condition, and replacing fresh tumor organoid culture solution every 3 days.

The method can further comprise the step of adding 0.125-1.25 g/L trypsin solution for digestion at 37 ℃ for 30 minutes to dissolve the hydrogel after the culture solution on the hydrogel is sucked and removed after the step (A4).

In a second aspect, the invention claims a kit for culturing a tumor organoid.

The kit for culturing tumor organoids claimed in the present invention comprises the following a1) -a 3):

a1) raw materials for preparing hydrogel: a hydrogel matrix, a coagulant, and a protein crosslinker;

a2) a cell filter for loading the hydrogel; the pore size of the cell filter is 10-70 μm (e.g., 40 μm);

a3) a centrifuge tube with a nozzle capable of fitting the cell filter (so that the cell filter is stuck at the nozzle, as shown in fig. 1).

Wherein the hydrogel matrix can be selected from any one of the following: isolated sheep plasma or other mammalian or human plasma, alginate, gelatin, hyaluronic acid, agarose, laminin, collagen, fibrin, Matrigel, and other synthetic or natural materials.

Wherein the coagulant is one or more of inorganic salt or/and organic salt containing calcium ions.

Further, the coagulant may be selected from any one or more of: calcium chloride, calcium gluconate, calcium dihydrogen phosphate, calcium nitrate, calcium bicarbonate, calcium hydrogen sulfate, calcium hydrogen sulfite and calcium hypochlorite.

In a particular embodiment of the invention, the accelerator is calcium chloride.

Wherein the protein cross-linking agent can be selected from any one or more of the following: glutaraldehyde, EDC, SMCC, DSS, DST, MBS, SPDP.

In a particular embodiment of the invention, the protein cross-linking agent is glutaraldehyde.

In a specific embodiment of the invention, the hydrogel is formed by taking isolated sheep plasma as a matrix and adding calcium chloride and glutaraldehyde; wherein the concentration of calcium chloride in the hydrogel is 3-6mmol/L (e.g. 4.5mmol/L), and the volume percentage of glutaraldehyde in the hydrogel is 0.1-0.5% (e.g. 0.25%).

Wherein the calcium chloride and the glutaraldehyde are added in the form of a 100 Xstock solution.

The kit may also contain a tumor organoid culture solution, as desired.

In a third aspect, the invention claims any of the following applications:

p1, the use of a kit as described in the second aspect above for culturing (in vitro culturing) a tumor organoid;

p2, the use of a kit as described in the second aspect hereinbefore for culturing (in vitro culturing) normal tissue organoids.

In the above aspects, the tumor may be a malignant tumor of epithelial tissue origin or a malignant tumor of mesenchymal tissue origin.

Further, the malignant tumor of epithelial tissue origin may be selected from any one of: lung cancer, breast cancer, stomach cancer, colorectal cancer, bladder cancer, kidney cancer, liver cancer, ovarian cancer, cervical cancer, and pancreatic cancer; the malignant tumor of mesenchymal tissue origin can be selected from any one of the following: chondrosarcoma and interstitial tumor.

The method of the invention has the following characteristics:

1. the sheep blood plasma is cheap and easy to obtain, and the culture cost is greatly reduced;

2. the low-concentration procoagulant and crosslinking reagents are jointly used, so that predominant proteins (albumin and globulin) in plasma form crosslinked protein, and the crosslinked protein and fibrin generated by procoagulant reaction cooperate to form a net-shaped porous loose structure. The prepared hydrogel has good water permeability;

3. the culture solution continuously and slowly permeates downwards through the loose porous structure of the hydrogel under the action of gravity, cells are fully supported by nutrition and take away metabolites in time, the culture success rate is improved, and the culture period of tumor organoids is shortened.

Drawings

FIG. 1 is a flow chart of the preparation of a permeable hydrogel scaffold.

Figure 2 is a microscopic structure of a permeable hydrogel scaffold.

FIG. 3 shows organoid culture effect of control group and permeable hydrogel scaffold group.

Figure 4 is a comparison of organoid volumes in control and permeable hydrogel scaffold groups.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The results shown in the following examples are all verified by repeated experiments.

Example 1 optimization of hydrogel preparation method

Calcium chloride and glutaraldehyde were added at different concentrations to sheep plasma (zhengzhou jiulong biologics ltd). Human liver cancer HepG2 cells (ATCC, USA) were cultured in two dimensions to logarithmic phase, and then digested with 0.25% (% represents g/100mL) trypsin (Sigma) to obtain single cell suspension at final concentration of 5X 10³The mixture is evenly mixed with the above groups (calcium chloride and glutaraldehyde with different concentrations are added into the sheep plasma) and then is spread in cell cultureThe plate wells were bottomed and each set was observed for clotting time. DMEM medium (Thermo Scientific) containing 10% (v/v) fetal bovine serum (Thermo Scientific) and 1% (m/v) penicillin/streptomycin was added to the gel with a setting time of 30 minutes or less. At 37 deg.C, 5% CO₂The state of each group of cells was observed after 3 days of culture under the conditions. The result shows that after 3-6mmol/L calcium chloride and 0.1-0.5% (v/v) glutaraldehyde are added into sheep plasma, hydrogel can be quickly formed in the plasma, the coagulation time is 5-10min, the states of all groups of cells are good, and HepG2 cells can grow into spherical three-dimensional structures in the gel. The optimum combination is 4.5mmol/L calcium chloride and 0.25% (v/v) glutaraldehyde.

Example 2 preparation of permeable hydrogel scaffolds

1. Preparing 100X calcium chloride storage liquid

Weighing 3.3294-6.6588 g of anhydrous calcium chloride, dissolving in 100ml of deionized water, wherein the concentration of the calcium chloride is 300-600mmol/L, and filtering and sterilizing by using a filter membrane with the aperture of 0.22 mu m.

2. Preparing 100 Xglutaraldehyde storage liquid

Measuring 10-50 mL of glutaraldehyde, adding deionized water to 100mL of glutaraldehyde, wherein the concentration of the glutaraldehyde is 10-50% (v/v), and filtering and sterilizing by using a filter membrane with the pore diameter of 0.22 mu m.

3. Sheep plasma: zhengzhou jiulong biological products, Inc.

4. Preparation of permeable hydrogel scaffolds

Isolating primary tumor cells from tumor tissue, counting the number of cells, adding a volume of sheep plasma to a final tumor cell concentration of 5X 10³and/mL. Then, according to the volume of the sheep plasma, 100 times of calcium chloride stock solution and 100 times of glutaraldehyde stock solution are added in a certain volume, so that the final concentration of glutaraldehyde is 0.25% (v/v), and the final concentration of calcium chloride is 4.5 mmol/L. After fully mixing, standing for 3min at room temperature (20-25 ℃) to make the suspension become viscous, after mixing uniformly again, slowly dripping the suspension to the bottom of a cell filter (the aperture is 40 mu m) to make the suspension completely fill the bottom of the cell filter, and the minimum volume of the suspension completely filling the bottom of the cell filter is 1 mL. Standing at 37 deg.C for 7min until the suspension solidifies to form a hydrogel. Adding organoid culture medium (organoid culture) on top of hydrogelLiquid volume: hydrogel volume 1: 1) the cell filter was placed horizontally in the mouth of a 50mL centrifuge tube, covered with a lid, and placed vertically at 37 ℃ in 5% CO₂Culturing under the condition. The permeable hydrogel scaffold preparation procedure is shown in figure 1. Under a microscope, the hydrogel was seen to form a loose porous structure between the filter pores of the cell filter (FIG. 2).

5. Evaluation of the permeation effect of the permeable hydrogel scaffold on the culture solution:

in order to detect whether the prepared permeable hydrogel scaffold can realize the function of permeating the culture solution, cell filters with the same specification are divided into two groups. Control group: the bottom filter screen is completely sealed by paraffin; experimental groups: the bottom screen is not sealed. Preparing hydrogel scaffold from two groups of cell filters by the method of step 4, adding culture solution with the same volume above the solidified hydrogel, placing the cell filters in the mouth of a 50mL centrifuge tube, covering the tube with a cover, and placing the tube at 37 deg.C and 5% CO₂Incubated under conditions for three days, and the volume of the culture broth (residual volume) above each group of hydrogels was measured. Permeation volume-control residual volume-experimental residual volume. Through detection, the prepared permeable hydrogel scaffold can realize the permeation function to a culture solution, and the permeation rate is about 300 mu L/day.

Example 3 evaluation of Effect of permeable hydrogel scaffolds on culturing tumor organoids

This example examined organoid culture effects of lung, ovarian and intestinal cancers on permeable hydrogel scaffolds.

1. Human tumor tissue samples were washed three times with pre-cooled Hank's balanced salt solution (containing 200U/mL penicillin, 200mg/mL streptomycin, and 0.5mg/mL amphotericin B, all from Sigma).

2. The tissue is cut to about 0.5mm by a sterile scissors³Small pieces of (a). The minced tissue was transferred to a 15mL centrifuge tube and approximately 10mL of pre-cooled Hank's Balanced salt solution was added. Aspirate several times up and down with a 10mL pipette.

3. Standing the centrifuge tube for 2-3 min to allow the tissue mass to settle to the bottom of the tube, and removing about 7.5mL of supernatant by suction. The washing step was repeated once.

4. After centrifugation at 200g for 5min, about 10mL of pre-warmed tissue digest (formulation: DMEM/F12 medium containing 0.001% DNase (Sigma), 1mg/mL collagenase/dispase (Roche), 200U/mL penicillin, 200mg/mL streptomycin, and 0.5mg/mL amphotericin B) was added to the 100mm dish after all the supernatant was aspirated. Digesting at 37 deg.C for 45-60 min. During which it oscillates several times.

5. The suspension was repeatedly aspirated several times through a 10mL pipette and filtered through a 70 μm pore size cell filter. The filtrate was centrifuged at 200g for 5 min.

6. Cell viability was assessed by 0.4% trypan blue staining and the viable cell fraction should be above 95%. The number of cells was counted using a cytometer.

7. Adding sheep plasma according to the number of cells to make the final concentration of the cells 5X 10³And adding a certain volume of 100 multiplied by calcium chloride storage solution and 100 multiplied by glutaraldehyde storage solution into the mixture to prepare a three-dimensional culture hydrogel solution, wherein the final concentration of glutaraldehyde is 025% (v/v), and the final concentration of calcium chloride is 4.5 mmol/L.

Control group: the three-dimensional culture hydrogel solution is fully and uniformly mixed and then is paved on the bottom of a six-hole cell culture plate hole (2 mL/hole),

permeable hydrogel scaffold group: the three-dimensional culture hydrogel solution is fully mixed, then is kept stand for 3min at room temperature to make the suspension become viscous, and is uniformly mixed again, and then 2mL of the suspension is sucked and completely paved on the bottom of a cell filter (Corning) with the aperture of 40 mu m.

Standing at 37 deg.C for 7min until the suspension solidifies to form a hydrogel. 2mL organoid culture was added on top of the hydrogel, the cell filter was placed in the mouth of a 50mL centrifuge tube as shown in FIG. 1, and the cell filter and control were placed at 37 ℃ in 5% CO₂Culturing under conditions (permeable hydrogel scaffold set centrifuge tubes placed vertically). Fresh organoid culture medium was replaced every 3 days. The components of the culture solution of various tumor organoids are as follows:

lung cancer organoid culture fluid: DMEM/F12(Thermo Scientific) was supplemented with 20ng/mL bFGF (basic fibroblast growth factor) (Invitrogen), 50ng/mL human EGF (epidermal growth factor) (Invitrogen), 1 XN 2(Invitrogen), 1 XB 27(Invitrogen), 10. mu.M ROCK inhibitor (Enzo Life Sciences) and 1 XPenicillin/Streptomyces (Penicillin-Streptomycin) (Gibco). The concentrations of the above substances are the final concentrations in the culture medium.

Ovarian cancer organoid culture fluid: advanced DMEM/F12(Gibco) supplemented with 1 XGlutamax (Gibco), 10mM HEPES (Gibco), 1 XPenicillin/Streptomyces (Penicillin-Streptomycin) (Gibco), 50. mu.g/mL Primocin (Gibco), 1 XB 27(Invitrogen), 5mM Nicotinamide (Nicotinamide) (Sigma), 1.25mM N-acetyl cysteine (acetylcysteine) (Sigma), 250ng/mL R-spondin3(Invitrogen), 5nM Heregulin beta-1 (Invitrogen), 100ng/mL Noggin (PeproTech), 20ng/mL FGF-10 (fibroblast growth factor-10) (PeproTech), 5ng/mL FGF-7 (fibroblast growth factor-7) (Invitrogen), 5ng/mL EGF (epidermal growth factor) (Invitrogen), 500nM A83-01(Invitrogen), 500nM SB202190(Invitrogen), 5. mu. M Y-27632 (Sigma). The concentrations of the above substances are the final concentrations in the culture medium.

Intestinal cancer organoid culture solution: advanced DMEM/F12(Gibco) was supplemented with 10nM Gastrin (Gastrin) (Sigma), 1 XB 27(Invitrogen), 1 XN 2(Invitrogen), 1mM N-acetyl cysteine (acetylcysteine) (Sigma), 1 Xglutamax (thermo scientific), 5% FBS (fetal bovine serum) (Gibco), 100. mu.g/mL gentamicin (gentamicin) (Solarbio), 1.25. mu.g/mL amphotericin B (amphotericin B) (Gibco), 100. mu.g/mL mycopricin (Invivogen), 10. mu.M SB202190(Sigma), 10mM Y-27632(Sigma), 50ng/mL EGF (epidermal growth factor) (Invitrogen), 5/mL FGF bbFGF (basic fibroblast growth factor) (Invitrogen). The concentrations of the above substances are the final concentrations in the culture medium.

8. After 3 weeks of culture, the control group and the permeable hydrogel scaffold group formed three-dimensional structures with different shapes and sizes (fig. 3). The volumes of the two groups of organoids are measured by Novisight 3D analysis software, and the result is shown in figure 4, after 3 weeks of culture, the average volume of the organoids of the permeable hydrogel stent group is obviously larger than that of the control group, which indicates that the culture period of the tumor organoids can be obviously shortened by adopting the permeable hydrogel method.

9. Subculturing: and after culturing for 3-4 weeks, removing the organoid culture solution on the hydrogel, adding 2mL of 0.25% (% represents g/100mL) trypsin, repeatedly blowing and sucking the hydrogel by a pipette gun, digesting for 30min at 37 ℃, and oscillating for several times during the period to ensure that the hydrogel is completely enzymolyzed into a solution, thereby releasing the organoids.

10. After centrifugation at 200g for 5min, all the supernatant was aspirated, and the digestion solution (formula: DMEM/F12 medium containing 0.001% DNase (Sigma), 1mg/mL collagenase/dispase (Roche), 200U/mL penicillin, 200mg/mL streptomycin) was added to a 100mm dish. Digesting at 37 deg.C for 45-60 min. During which the tumor organoids are digested into single cell suspensions by shaking several times.

11. Centrifuging at 200g for 5min, sucking up all supernatant, and performing cell subculture according to the ratio of 1 to 3. The operation method is shown in the step 7 to the step 8.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Claims

1. A method of culturing a tumor organoid, comprising the steps of:

(A1) mixing tumor cells to be cultured with hydrogel matrix to make the final concentration of cells be (5-10) × 10³/mL；

(A3) placing the hydrogel solution containing tumor cells at 20-25 ℃ until the hydrogel solution becomes viscous, adding the hydrogel solution into the bottom of a cell filter with the pore diameter of 10-70 mu m, flatly placing and standing at 37 ℃ until the hydrogel solution containing tumor cells is solidified, and adding a tumor organoid culture solution above the hydrogel;

(A4) horizontally placing the cell filter (A3) filled with the hydrogel and the culture solution in a nozzle of a centrifuge tube capable of being matched with the cell filter, and vertically placing the centrifuge tube for culturing to obtain the tumor organoid.

2. The method of claim 1, wherein: in the step (a1), the hydrogel matrix is selected from any one of the following: isolated sheep or other mammalian or human plasma, alginate, gelatin, hyaluronic acid, agarose, laminin, collagen, fibrin, Matrigel;

further, the hydrogel matrix is sheep plasma ex vivo.

3. The method according to claim 1 or 2, characterized in that: in the step (a2), the coagulant is any one or more of inorganic salt or/and organic salt containing calcium ions;

further, the coagulant is selected from any one or more of: calcium chloride, calcium gluconate, calcium dihydrogen phosphate, calcium nitrate, calcium bicarbonate, calcium hydrogen sulfate, calcium hydrogen sulfite, and calcium hypochlorite;

still further, the coagulant is calcium chloride;

more specifically, the concentration of the calcium chloride in the hydrogel solution is 3-6 mmol/L;

and/or

In step (a2), the protein cross-linking agent is selected from any one or more of: glutaraldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide ester of 4- (N-maleimidomethyl) cyclohexanecarboxylic acid, disuccinimidyl suberate, disuccinimidyl tartrate, m-maleimidobenzoyl-N-hydroxysuccinimide ester, succinimide 3- (2-pyridyldithio) -propionate;

further, the protein cross-linking agent is glutaraldehyde;

still further, the glutaraldehyde is present in the hydrogel solution in an amount of 0.1 to 0.5% by volume.

4. A method according to any one of claims 1-3, characterized in that: in the step (A3), the hydrogel solution containing the tumor cells is placed at room temperature for 3-5min and then added to the bottom of a cell filter with the pore diameter of 10-70 μm;

and/or

In the step (A3), after the hydrogel solution containing the tumor cells is added to the bottom of the cell filter, keeping flat and standing at 37 ℃ for 5-7 min;

and/or

In step (a3), the volume of the tumor organoid culture fluid added over the hydrogel is equal to the volume of the hydrogel.

5. The method according to any one of claims 1-4, wherein: the method further comprises the step of adding 0.125-1.25 g/L trypsin solution for digestion at 37 ℃ for 30 minutes to dissolve the hydrogel after the culture solution on the hydrogel is sucked and removed after the step (A4).

6. A kit for culturing tumor organoids, comprising: the kit contains the following a1) -a 3):

a2) a cell filter for loading the hydrogel; the pore size of the cell filter is 10-70 μm;

a3) a centrifuge tube having a nozzle capable of fitting the cell filter.

7. The kit of claim 6, wherein: the hydrogel matrix is selected from any one of the following: isolated sheep or other mammalian or human plasma, alginate, gelatin, hyaluronic acid, agarose, laminin, collagen, fibrin, Matrigel;

further, the hydrogel matrix is in vitro sheep plasma;

and/or

The coagulant is any one or more of inorganic salt or/and organic salt containing calcium ions;

still further, the coagulant is calcium chloride;

and/or

The protein cross-linking agent is selected from any one or more of the following: glutaraldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide ester of 4- (N-maleimidomethyl) cyclohexanecarboxylic acid, disuccinimidyl suberate, disuccinimidyl tartrate, m-maleimidobenzoyl-N-hydroxysuccinimide ester, succinimide 3- (2-pyridyldithio) -propionate;

further, the protein cross-linking agent is glutaraldehyde;

and/or

The hydrogel is formed by taking in-vitro sheep plasma as a matrix and adding calcium chloride and glutaraldehyde; wherein, the concentration of calcium chloride in the hydrogel is 3-6mmol/L, and the volume percentage of glutaraldehyde in the hydrogel is 0.1-0.5%.

8. The kit of claim 6 or 7, wherein: the kit also contains a tumor organoid culture solution.

9. Any of the following applications:

use of P1 or the kit of any one of claims 6 to 8 in culturing a tumor organoid;

use of P2, the kit of any one of claims 6 to 8, in culturing a normal tissue organoid.

10. The method or kit or use according to any one of claims 1 to 9, wherein: the tumor organoid is an organoid of a malignant tumor derived from epithelial tissue or an organoid of a malignant tumor derived from mesenchymal tissue;

further, the malignant tumor of epithelial tissue origin is selected from any one of: lung cancer, breast cancer, stomach cancer, colorectal cancer, bladder cancer, kidney cancer, liver cancer, ovarian cancer, cervical cancer, and pancreatic cancer; the malignant tumor of mesenchymal tissue origin is selected from any one of the following: chondrosarcoma and interstitial tumor.