CN118028238A - Human colorectal cancer organoid resuscitation liquid, culture liquid and resuscitation method - Google Patents

Abstract

The invention discloses a human colorectal cancer organoid resuscitation liquid, a culture liquid and a resuscitation method, which belong to the organoid resuscitation technical field, wherein the resuscitation liquid contains Z-VAD-FMK with the concentration range of 0-100 mu M and Necrostatin-1 with the concentration range of 0-40 mu M, and the concentration ranges from 0 mu M to 0 mu M at different times; when the resuscitation fluid provided by the invention is used for resuscitating colorectal cancer organoids, the resuscitation fluid can obviously improve the growth vigor of the human colorectal cancer organoids, shorten the resuscitating process, furthest improve the resuscitating efficiency of the human colorectal cancer organoids, and simultaneously does not obviously change the original biological characteristics.

Description

A human colorectal cancer organoid resuscitation fluid, culture fluid and resuscitation method

Technical Field

The present invention relates to the technical field of organoid resuscitation, and in particular to a human colorectal cancer organoid resuscitation fluid, culture medium and resuscitation method.

Background technique

Colorectal cancer is a malignant tumor of the digestive system with high morbidity and mortality, and has biological characteristics such as high recurrence, high metastasis and low survival. However, there are still many challenges in its early diagnosis and clinical treatment. Therefore, a large number of in vitro studies on colorectal cancer are urgently needed to further reveal its pathogenesis and develop new drug treatment targets, thereby reducing its harm to human health.

Organoids are a new in vitro cell culture technology that is a miniaturized and simplified version of an organ, or an "ex vivo" mini-organ that can provide a microenvironment similar to that in vivo. Organoids have the characteristics of being passable, resuscitable, gene-editable, and having stable genetic information. Organoid models can well simulate the microenvironment of cells in the body and have obvious advantages in constructing research models with physiological functions in vitro. At present, the application directions of organoids cover disease models, personalized medicine, new drug development, regenerative medicine and other aspects.

Human colorectal cancer organoids are organoids cultured from fresh samples of colorectal cancer from clinical patients. Their sources are very valuable and cannot be obtained repeatedly. Therefore, they need to be cryopreserved during the research process. Although they can be frozen and revived, their resuscitation efficiency is very low under conventional methods, and the resuscitation cycle generally exceeds 14 days. CN117625537A-New culture medium for culturing colorectal cancer organoids discloses a culture medium for culturing colorectal cancer organoids, which contains specific added factors including insulin growth factor and Gremlin1, ALK 5 inhibitor, ROCK inhibitor, MAPK inhibitor, R Spondin, B27 and Gastrin1, but the scheme has too many types of additives, high cost, and complex experimental design. At the same time, ALK inhibitors and MAPK inhibitors may change the original biological characteristics of biological samples, thereby destroying the genetic stability of organoids.

Therefore, when resuscitating human colorectal cancer organoids, there is an urgent need for a technology that can enhance their growth vitality, increase survival rate, shorten the resuscitation cycle, and at the same time not change their genetic stability, thereby providing researchers and clinicians with valuable organoid resources.

Summary of the invention

The purpose of the present invention is to provide a human colorectal cancer organoid resuscitation fluid to solve the above problems.

In order to achieve the above-mentioned purpose, the technical solution adopted by the present invention is as follows: a human colorectal cancer organoid resuscitation fluid, wherein the resuscitation fluid contains Z-VAD-FMK and Necrostatin-1.

The inventors of the present application have obtained two functional small molecules, Z-VAD-FMK and Necrostatin-1, through a large number of experiments, which can solve the above problems, wherein:

Z-VAD-FMK (Z-VAD(OH)-FMK) is a pan-apoptosis inhibitor with the chemical formula:

;

Z-VAD-FMK does not inhibit ubiquitin C-terminal hydrolase L1 (UCHL1) activity, even at concentrations as high as 440μM. The pan-apoptosis inhibitor Z-VAD-FMK can very significantly inhibit apoptosis induced by most apoptosis inducers at a final concentration of 20μM. The common effective concentration range for the pan-apoptosis inhibitor Z-VAD-FMK to inhibit apoptosis is 5-100μM. Z-VAD-FMK is a cell-permeable, irreversible pan-caspase inhibitor. Inhibits caspase processing and apoptosis induction in tumor cells in vitro. Once inside the cell, endogenous esterase activity hydrolyzes the methyl group to form the biologically active form. The effective concentration range of Z-VAD-FMK described in the present invention is 0-100μM.

Necrostatin-1 (Nec-1) is an effective necroptosis inhibitor that can penetrate the blood-brain barrier. Its chemical structure is:

;

EC50 in Jurkat cells is 490nM. Necrostatin-1 inhibits RIP1 kinase (EC50=182 nM). Necrostatin-1 (Nec-1) is also an (IDO) inhibitor. The effective concentration range of Necrostatin-1 described in the present invention is 0-40μM.

As a preferred technical solution, the concentration range of Z-VAD-FMK in the resuscitation solution is 0-100 μM, the concentration range of Necrostatin-1 is 0-40 μM, and they are not 0 μM at the same time.

The concentration ranges mentioned refer to the culture range in growth medium.

As a further preferred technical solution, the concentration range of Z-VAD-FMK in the resuscitation solution is 3.125-100 μM, and the concentration range of Necrostatin-1 is 1.25-40 μM.

As a further preferred technical solution, the concentration of Z-VAD-FMK in the resuscitation solution is 25 μM, and the concentration of Necrostatin-1 is 10 μM.

As a further preferred technical solution, a human colorectal cancer organoid resuscitation fluid is characterized in that human colorectal cancer organoid growth culture fluid is used as a basal fluid, and the basal fluid components include Advanced DMEM/F12 cell culture fluid, L-WRN culture medium, 10 vol.% FBS, 10 mM HEPPS, 2 mM Gluta-max, 500 U/mL penicillin-streptomycin, 1× N-2 additive, 1× B27 additive and 10 μM Y27632, and Z-VAD-FMK and Necrostatin-1 as the mother liquor of human colorectal cancer organoid resuscitation fluid are added to the basal fluid to obtain human colorectal cancer organoid resuscitation fluid.

The second object of the present invention is to provide a method for preparing the above-mentioned human colorectal cancer organoid resuscitation fluid, comprising the following steps:

(1) Preparation of human colorectal cancer organoid resuscitation solution stock solution: Z-VAD-FMK and Necrostatin-1 were added to DMSO to completely dissolve them, and then filtered to sterilize to obtain the resuscitation solution stock solution;

(2) Preparation of human colorectal cancer organoid growth culture medium: its components include Advanced DMEM/F12 cell culture medium, L-WRN medium, 10 vol.% FBS, 10 mM HEPPS, 2 mM Gluta-max, 500 U/mL penicillin-streptomycin, 1× N-2 supplement, 1× B27 supplement, and 10 μM Y27632;

(3) Preparation of human colorectal cancer organoid resuscitation solution: Add the human colorectal cancer organoid resuscitation solution stock solution obtained in step (1) to the human colorectal cancer organoid growth culture medium in step (2) to dilute and mix well.

The third object of the present invention is to provide a method for resuscitating and culturing colorectal cancer organoid cells using the above-mentioned human colorectal cancer organoid resuscitation fluid, wherein the frozen human colorectal cancer organoid cells are resuscitated using the human colorectal cancer organoid resuscitation fluid, and the method comprises the following steps:

(1) Open the water bath in advance, put the matrix gel into the refrigerator to melt, put it on ice before the experiment, pre-cool the pipette tips needed for the experiment, and pre-heat the required culture dishes in the incubator in advance;

(2) Take out the frozen human colorectal cancer organoid cells and immediately transfer them to a water bath to thaw, shaking them while thawing;

(3) Take a centrifuge tube and add pre-cooled PBS, add the thawed cell suspension into the centrifuge tube, mix gently and centrifuge;

(4) Discard the supernatant and add organoid growth medium to gently suspend the cells;

(5) Mix the cell suspension with the matrix gel, drop the mixture into a culture dish, let it stand, and place the culture dish in an incubator to solidify the matrix gel;

(6) taking out the culture dish, adding the prepared human colorectal cancer organoid resuscitation solution into the culture dish, and placing it in an incubator for culture;

(7) After treatment, replace the human colorectal cancer organoid growth medium and continue culturing, changing the medium every 3-4 days.

Compared with the prior art, the advantages of the present invention are that when the resuscitation fluid of the present invention is used to resuscitate colorectal cancer organoids, the resuscitation fluid can significantly improve the growth vitality of human colorectal cancer organoids, shorten their resuscitation process, and maximize the resuscitation efficiency of human colorectal cancer organoids. At the same time, it is low-cost and easy to prepare, and does not significantly change their original biological characteristics.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a general picture of the recovery of human colorectal cancer organoids after treatment with different resuscitation fluids;

Figure 2 shows the statistics of the number of human colorectal cancer organoids after treatment with different resuscitation fluids;

Figure 3 shows the diameter statistics of human colorectal cancer organoids after treatment with different resuscitation fluids;

Figure 4 is a general picture of organoid recovery after treatment with the recovery solution Z-VAD (25μM) + Nec-1 (10μM);

Figure 5 is a statistical graph of the number of organoids after treatment with the resuscitation solution Z-VAD (25μM) + Nec-1 (10μM);

Figure 6 is a statistical graph of organoid diameters after treatment with resuscitation solution Z-VAD (25μM) + Nec-1 (10μM);

FIG7 is a general picture of organoids treated with Z-VAD (25 μM) or Nec-1 (10 μM) alone and the combined treatment of the two after 48 hours of recovery;

Figure 8 shows Ki67 immunofluorescence staining photos of organoids under the no resuscitation fluid group and the optimal resuscitation fluid conditions.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings.

The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental reagents used are purchased from conventional reagent manufacturers unless otherwise specified. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in the field or the product instructions are used. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

The following embodiments involve:

1. Experimental environment: Operation in a biological safety cabinet in a laboratory under a GMP environment;

2. Experimental reagents: All reagents used were purchased from the market, as follows:

Reagent name Buying a company Z-VAD-FMK MCE Necrostatin-1 MCE Advanced DMEM/F12 Gibco FBS (fetal bovine serum μM) BI Company HEPES Gibco Gluta-max Gibco PS (Penicillin-Streptomycin) Shanghai Biyuntian Company N-2 Additive Gibco B27 Additive Gibco Y-27632 Sigma Matrigel Corning Ki67 antibody Abcam AlexFluo594 goat anti-rabbit secondary antibody Abcam

;

3. Instruments and equipment: _CO2 incubator, biological safety cabinet, microplate reader, centrifuge, electronic balance, 96-well plate.

Embodiment 1:

A human colorectal cancer organoid resuscitation solution, mainly comprising Z-VAD-FMK with an effective concentration range of 0-100 μM and Necrostatin-1 with an effective concentration range of 0-40 μM, grouped as follows;

a.100μM Z-VAD-FMK+40μM Necrostatin-1,

b.50μM Z-VAD-FMK+20μM Necrostatin-1,

c.25μM Z-VAD-FMK+10μM Necrostatin-1,

d.12.5μM Z-VAD-FMK+5μM Necrostatin-1,

e.6.25μM Z-VAD-FMK+2.5μM Necrostatin-1,

f.3.125μM Z-VAD-FMK+1.25μM Necrostatin-1,

g.0μM Z-VAD-FMK+0μM Necrostatin-1,

The remaining ingredients are: Advanced DMEM/F12 cell culture medium, L-WRN medium, 10% FBS, 10mM HEPPS, 2mM Gluta-max, 500U/mL penicillin-streptomycin, 1× N-2 supplement, 1× B27 supplement, and 10μM Y27632.

The “human colorectal cancer organoid growth medium” mentioned below refers to the “other components” that do not contain Z-VAD-FMK and Necrostatin-1.

According to an embodiment of the present invention, the final concentration of HEPES in the culture medium is 10 mM.

According to an embodiment of the present invention, the final concentration of Gluta-max in the culture medium is 2 mM.

According to an embodiment of the present invention, the final concentration of penicillin in the culture medium is 500 U/ml.

According to an embodiment of the present invention, the final concentration of streptomycin in the culture medium is 500 U/ml.

According to an embodiment of the present invention, the final concentration of Y27632 in the culture medium is 10 μM.

It should be noted that "the final concentration of the N-2 additive and the B27 additive in the culture medium is 1×" means that the concentration of the N2 and B27 products is 100×. When preparing the culture medium, the final concentration of N2 and B27 in the culture medium is diluted to 1×.

Specifically, in this embodiment, the cells are human colorectal cancer organoid cells.

The specific steps include:

(1) Turn on the water bath and adjust the temperature to 37°C. Dissolve the Matrigel on ice in advance.

(2) Take out the frozen human colorectal cancer organoids, immerse them in a 37°C water bath, and shake them from time to time to thaw them as quickly as possible;

(3) Add 5 ml of human colorectal cancer organoid growth medium to a 15 ml centrifuge tube, add the thawed cell suspension to the centrifuge tube, mix gently, and then centrifuge (1500 rpm, 5 min);

(4) Preheat the 96-well plate in an incubator for 10 minutes; discard the supernatant after centrifugation in step (3) to obtain a cell suspension, mix the cell suspension with Matrigel at a volume ratio of 1:9 to avoid the generation of bubbles, drop 5 μl of the mixture into the 96-well plate, and place the 96-well plate in an incubator for about 15 minutes to allow the Matrigel to solidify;

(5) Take out the 96-well plate, add the human colorectal cancer organoid resuscitation solution prepared according to the grouping, and place it in an incubator at 37°C and 5 vol.% _CO2 for continued cultivation;

(6) After different periods of culture in the organoid resuscitation medium, the human colorectal cancer organoid growth medium was replaced for continued culture. During the resuscitation period, the growth of the organoids was regularly monitored and images were collected. All images in this article were collected on the 10th day after the organoids were resuscitated, and the results are shown in the figure.

(7) Use ImageJ software to count the number and size of organoids;

(8) Use GraphPad software to calculate the results and plot the graph.

As shown in Figures 1, 2 and 3, when human colorectal cancer organoids were revived, different concentrations of Z-VAD-FMK+Necrostatin-1 were added to the growth culture medium of human colorectal cancer organoids. In the range of Z-VAD-FMK (0-25 μM) + Necrostatin-1 (0-10 μM), as the concentration increased, the number and volume of organoids increased. When the concentration was Z-VAD-FMK (25 μM) + Necrostatin-1 (10 μM), the organoid growth ability was the strongest. In the range of Z-VAD-FMK (25-100 μM) + Necrostatin-1 (10 -40 μM), the number and volume of organoids showed a gentle trend. Therefore, Z-VAD-FMK (25 μM) + Necrostatin-1 (10 μM) was selected as the optimal concentration of the resuscitation solution.

As shown in Figures 4, 5 and 6, when human colorectal cancer organoids were revived, the organoids were treated with Z-VAD-FMK (25 μM) + Necrostatin-1 (10 μM) for different time periods. Within 0-48 hours, the efficiency of organoid resuscitation increased with increasing exposure time, and the growth of organoids tended to be flat within 48-168 hours. Therefore, Z-VAD-FMK (25 μM) + Necrostatin-1 (10 μM) treatment for 48 hours is the best way to resuscitate human colorectal cancer.

In view of the above optimal drug concentration and duration of action, the inventors compared the effects of resuscitation solutions containing a single drug and a combination of drugs on the resuscitation of human colorectal cancer organoids. As shown in Figure 7, when human colorectal cancer organoids were resuscitated, the number of organoids treated with Z-VAD-FMK (25 μM) or Necrostatin-1 (10 μM) alone for 48 hours was significantly lower than that treated with Z-VAD-FMK (25 μM) + Necrostatin-1 (10 μM) for 48 hours. The above shows that the combination of the two in an appropriate ratio achieves a synergistic technical effect.

Embodiment 2:

This example provides an immunofluorescence staining method for human colorectal cancer organoids. In this example, rabbit anti-Ki67 antibody (1:200), AlexFluo594 goat anti-rabbit secondary antibody (1:500),

The specific steps include:

(1) Organoid fixation: Stop culturing organoids 15-20 days after recovery, discard the culture medium, wash twice with PBS, add cell recovery solution on ice to digest and release organoids, remove the recovery solution after the organoids naturally settle, and add 4 vol.% paraformaldehyde to fix for 30 min;

(2) Dehydration: The fixed organoids were resuspended in 2 vol.% agarose (heated and melted), and then dehydrated in a tissue dehydrator;

(3) Embedding and sectioning: The dehydrated organoids are embedded in a tissue embedding machine and sectioned on a microtome with a thickness of about 5 μm.

(4) Baking: Take the paraffin-embedded slices and bake them in an oven for about 2 hours;

(5) Dewaxing and hydrating paraffin-embedded sections: For paraffin-embedded sections, place the sections in xylene I for 15 min, xylene II for 15 min, 100% anhydrous ethanol I for 10 min, 100% anhydrous ethanol II for 5 min, 90 vol.% alcohol for 5 min, 80 vol.% alcohol for 5 min, and rinse with running water for 5 min;

(6) Antigen repair: Use the EDTA antigen repair method. Heat the dewaxed and hydrated slides in (5) until the repair solution bubbles and keep it for 15 minutes. During this process, prevent excessive evaporation of the buffer and do not dry the slides. After the repair is completed, cool naturally and rinse with running water for 10 minutes;

(7) Blocking: Cover the tissue evenly with the blocking solution and block for 45-60 minutes at room temperature;

(8) Incubation with primary antibody: dilute the target antibody (1:200) with blocking solution, add antibody to cover the tissue, incubate overnight at 4°C in a humidified chamber, incubate with primary antibody for about 24 hours, wash the sections with PBS for 5 minutes on a shaker, three times;

(9) Incubation with secondary antibody: Add secondary antibody of the same species as the primary antibody to cover the tissue, incubate at room temperature in the dark for 40-60 min, and wash with PBS on a shaker three times, 5 min each time;

(10) Unbound secondary antibody was washed away and DAPI was counterstained. The sections were sealed with anti-fluorescence quenching sealing medium and photographed using the Zeiss 3D light sectioning imaging system.

The results are shown in Figure 8. The Ki67 immunofluorescence results showed that compared with the group without resuscitation fluid, after 48 hours of action of the resuscitation fluid Z-VAD-FMK (25 μM) + Necrostatin-1 (10 μM), there was no significant change in the proliferation ability of the organoids after resuscitation. The resuscitation fluid did not change the biological genetic stability of the organoids. This shows that this resuscitation fluid improves the efficiency of organoid resuscitation without affecting the original biological genetic characteristics of the organoids.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims (7)

1. A human colorectal cancer organoid resuscitation fluid, characterized in that the resuscitation fluid contains Z-VAD-FMK and Necrostatin-1.

2. The human colorectal cancer organoid resuscitation fluid according to claim 1, wherein the concentration of Z-VAD-FMK in said resuscitation fluid is in the range of 0-100 μΜ, and the concentration of Necrostatin-1 is in the range of 0-40 μΜ, and not simultaneously 0 μΜ.

3. The human colorectal cancer organoid resuscitation fluid according to claim 2, wherein said concentration of Z-VAD-FMK in said resuscitation fluid is in the range of 3.125-100 μΜ and said concentration of Necrostatin-1 is in the range of 1.25-40 μΜ.

4. The human colorectal cancer organoid resuscitation fluid according to claim 3, wherein said resuscitation fluid has a concentration of Z-VAD-FMK of 25 μΜ and a concentration of Necrostatin-1 of 10 μΜ.

5. The human colorectal cancer organoid resuscitation fluid according to claim 1, characterized in that a human colorectal cancer organoid growth culture fluid is used as a base fluid, said base fluid comprises ADVANCED DMEM/F12 cell culture fluid, L-WRN medium, 10vol.% FBS, 10mM HEPPS, 2mM Gluta-max, 500U/mL penicillin-streptomycin, 1 xn-2 additive, 1 xb 27 additive and 10 μ M Y27632, and Z-VAD-FMK and Necrostatin-1 as human colorectal cancer organoid resuscitation fluid mother fluids are added to said base fluid, thus obtaining human colorectal cancer organoid resuscitation fluid.

6. A method of preparing a human colorectal cancer organoid resuscitation fluid according to any one of claims 1 to 5, comprising the steps of:

(1) Preparing human colorectal cancer organoid resuscitation fluid mother liquor: adding DMSO into Z-VAD-FMK and Necrostatin-1 to dissolve the two completely, and filtering and sterilizing by a filter to obtain a resuscitation fluid mother liquor;

(2) Preparing a human colorectal cancer organoid growth culture solution: the components of the kit comprise ADVANCED DMEM/F12 cell culture solution, L-WRN medium, 10vol.% FBS, 10mM HEPS, 2mM Gluta-max, 500U/mL penicillin-streptomycin, 1 XN-2 additive, 1 XB 27 additive and 10 mu M Y27632;

(3) Preparing human colorectal cancer organoid resuscitation fluid: adding the human colorectal cancer organoid resuscitation fluid mother fluid obtained in the step (1) into the human colorectal cancer organoid growth culture fluid obtained in the step (2) for dilution, and uniformly mixing to obtain the human colorectal cancer organoid resuscitation fluid.

7. A method for resuscitating and culturing human colorectal cancer organoid cells using the human colorectal cancer organoid resuscitation fluid according to any one of claims 1 to 5, characterized in that the human colorectal cancer organoid cells that have been frozen are resuscitated using the human colorectal cancer organoid resuscitation fluid, the method comprising the steps of:

(1) Opening a water bath in advance, putting matrigel into a refrigerator in advance to melt, putting the matrigel on ice before experiments, pre-cooling gun heads required by the experiments in advance, and putting a required culture dish into an incubator in advance to be preheated;

(2) Taking out the frozen human colorectal cancer organoid cells, immediately transferring the frozen human colorectal cancer organoid cells into a water bath kettle for thawing, and shaking while thawing;

(3) Adding precooled PBS into a centrifuge tube, adding the unfrozen cell suspension into the centrifuge tube, lightly mixing and centrifuging;

(4) Removing the supernatant, and adding organoid growth culture solution to lightly suspend cells;

(5) Mixing the cell suspension with matrigel, dripping the mixture into a culture dish after uniform mixing, standing, and placing the culture dish into an incubator to solidify the matrigel;

(6) Taking out the culture dish, adding the prepared human colorectal cancer organoid resuscitation liquid into the culture dish, and placing the culture dish into an incubator for culture;

(7) After the treatment is finished, the culture solution is changed for the growth of the human colorectal cancer organoid to continue to be cultured, and the culture solution is changed for one time for 3-4 days.