CN118028238A - Human colorectal cancer organoid resuscitation liquid, culture liquid and resuscitation method - Google Patents
Human colorectal cancer organoid resuscitation liquid, culture liquid and resuscitation method Download PDFInfo
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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
Technical Field
The invention relates to the technical field of organoid resuscitation, in particular to a human colorectal cancer organoid resuscitation liquid, a culture liquid and a resuscitation method.
Background
Colorectal cancer is a digestive system malignant tumor with high morbidity and mortality, has biological characteristics of high recurrence, high metastasis, low survival rate and the like, and has many challenges in early diagnosis, clinical treatment and the like at present, so that a great deal of in vitro research on colorectal cancer is needed to further reveal the pathogenesis of the colorectal cancer and develop new drug treatment targets, thereby reducing the harm degree of the colorectal cancer to human health.
Organoids are a new cell culture technique in vitro, which is a miniaturized and simplified version of an organ, or "ex vivo" small organ, that provides a microenvironment that is comparable to that in vivo. The organoid has the characteristics of passaging, resuscitatability, gene editing, stable genetic information and the like, and the organoid model can well simulate the microenvironment of cells in vivo and has obvious advantages for constructing a research model with physiological functions in vitro. The current organoid application direction covers a plurality of links such as disease models, individuation medicine, new medicine research and development, regeneration medicine and the like.
The human colorectal cancer organoids are organoids obtained by culturing fresh colorectal cancer samples of clinical patients, and have valuable sources and unrepeatable acquisition characteristics, so that the organoids need to be frozen for seed preservation in the research process, and the organoids can be frozen for resuscitation, but the resuscitation efficiency is very low under the conventional method, and the resuscitation period is generally more than 14 days. CN117625537 a-a novel medium for culturing colorectal cancer organoids discloses a medium for culturing colorectal cancer organoids, which contains specific additives including insulin growth factor and Gremlin1, ALK 5 inhibitors, ROCK inhibitors, MAPK inhibitors, R Spondin, B27 and Gastrin1, but the scheme is too many in types, costly, complex in experimental design, and at the same time ALK inhibitors and MAPK inhibitors may alter the original biological properties of the biological sample, thereby destroying the genetic stability of organoids.
Therefore, in the resuscitation of human colorectal cancer organoids, there is a need for a technique that can increase the growth vigor, increase the survival rate, shorten the resuscitation cycle, and at the same time not change the genetic stability of the organoids, thereby providing valuable organoid resources for researchers and clinical workers.
Disclosure of Invention
The invention aims to provide a human colorectal cancer organoid resuscitation fluid for solving the problems.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a human colorectal cancer organoid resuscitation fluid, which comprises Z-VAD-FMK and Necrostatin-1.
The inventor of the present application can solve the above problems by obtaining two functional small molecules, Z-VAD-FMK and Necrostatin-1, through a large number of experiments, wherein:
Z-VAD-FMK (Z-VAD (OH) -FMK) is an inhibitor of ubiquity, and has the chemical structural formula:
；
Z-VAD-FMK does not inhibit ubiquitin C-terminal hydrolase L1 (UCHL 1) activity even at concentrations up to 440. Mu.M. The pan-apoptosis inhibitor Z-VAD-FMK can very significantly inhibit most apoptosis-inducing agents induced apoptosis at a final concentration of 20. Mu.M. The usual effective concentration range of the ubiquitously apoptosis inhibitor Z-VAD-FMK for inhibiting apoptosis is 5-100 mu M. Z-VAD-FMK is a cell permeable, irreversible inhibitor of ubiquitin caspase. Inhibition of caspase processing and apoptosis induction in tumor cells in vitro. Once in the cell, endogenous esterase activity hydrolyzes methyl groups to form a biologically active form. The effective concentration range of the Z-VAD-FMK is 0-100 mu M.
Necrostatin-1 (Nec-1) is a potent inhibitor of necrotic apoptosis across the blood brain barrier, and has the chemical structural formula:
；
The EC50 in Jurkat cells was 490nM. Necrostatin-1 inhibits RIP1 kinase (EC 50=182 nM). Necrostatin-1 (Nec-1) is also an (IDO) inhibitor. The effective concentration range of Necrostatin-1 is 0-40 mu M.
As a preferable technical scheme, the concentration range of Z-VAD-FMK in the resuscitation fluid is 0-100 mu M, and the concentration range of Necrostatin-1 is 0-40 mu M, and not 0 mu M at the same time.
The concentration range refers to a range of culture in a growth medium.
As a further preferable technical scheme, the concentration range of the Z-VAD-FMK in the resuscitation fluid is 3.125-100 mu M, and the concentration range of the Necrostatin-1 is 1.25-40 mu M
As a still further preferable embodiment, the concentration of Z-VAD-FMK in the resuscitation fluid is 25. Mu.M and the concentration of Necrostatin-1 is 10. Mu.M.
As a further preferable technical scheme, the human colorectal cancer organoid resuscitation fluid is characterized in that a human colorectal cancer organoid growth culture fluid is used as a base fluid, the base fluid comprises ADVANCED DMEM/F12 cell culture fluid, L-WRN culture 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, and Z-VAD-FMK and Necrostatin-1 serving as human colorectal cancer organoid resuscitation fluid mother liquor are added into the base fluid to obtain the human colorectal cancer organoid resuscitation fluid.
The second purpose of the invention is to provide a preparation method of the human colorectal cancer organoid resuscitation fluid, which comprises the following steps:
(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.
The invention also provides a method for recovering and culturing colorectal cancer organoid cells by using the humanized colorectal cancer organoid recovery liquid, wherein the method comprises the following 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.
Compared with the prior art, the invention has the advantages that: when the resuscitation fluid 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 is low in cost and easy to prepare, and the original biological characteristics of the human colorectal cancer organoids are not obviously changed.
Drawings
FIG. 1 is a general diagram of human colorectal cancer organoids resuscitated after treatment with different resuscitation fluids;
FIG. 2 is a graph showing statistics of the number of human colorectal cancer organoids after treatment with different resuscitation fluids;
FIG. 3 is a graph showing human colorectal cancer organoid diameter statistics after treatment with different resuscitation fluids;
FIG. 4 is a general diagram of organoid resuscitation after treatment with resuscitation fluid Z-VAD (25. Mu.M) +Nec-1 (10. Mu.M);
FIG. 5 is a statistical plot of organoid numbers following resuscitation fluid Z-VAD (25. Mu.M) +Nec-1 (10. Mu.M);
FIG. 6 is a statistical plot of organoid diameters after resuscitation fluid Z-VAD (25. Mu.M) +Nec-1 (10. Mu.M);
FIG. 7 is a schematic representation of organoids after resuscitation of Z-VAD (25. Mu.M) or Nec-1 (10. Mu.M) alone and in combination for 48 h;
FIG. 8 is a photograph of an immunofluorescence stain of organoid Ki67 under no resuscitation fluid and optimal resuscitation fluid conditions.
Detailed Description
The invention will be further described with reference to the accompanying drawings.
The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental reagents used, without any particular instruction, were purchased from conventional reagent manufacturers. The following examples are illustrative only and are not to be construed as limiting the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The following examples are presented:
1. Experimental environment: operating in a biological safety cabinet in a laboratory under a GMP environment;
2. Experimental reagent: the reagents used were all commercially available and were as follows:
| Reagent name | Purchasing company |
| Z-VAD-FMK | MCE Co Ltd |
| Necrostatin-1 | MCE Co Ltd |
| Advanced DMEM/F12 | Gibco Co Ltd |
| FBS（fetal bovine serμM） | BI Co Ltd |
| HEPES | Gibco Co Ltd |
| Gluta-max | Gibco Co Ltd |
| PS（Penicillin-Streptomycin ） | Shanghai Biyundian Co Ltd |
| N-2 additive | Gibco Co Ltd |
| B27 additive | Gibco Co Ltd |
| Y-27632 | Sigma Co |
| Matrigel | Corning Co Ltd |
| Ki67 antibodies | Abcam Co Ltd |
| AlexFluo594 goat anti-rabbit secondary antibody | Abcam Co Ltd |
；
3. Instrument and apparatus: the device comprises a CO 2 incubator, a biosafety cabinet, an enzyme-labeling instrument, a centrifuge, an electronic balance and a 96-well plate.
Example 1:
The human colorectal cancer organoid resuscitation fluid mainly comprises Z-VAD-FMK with the effective concentration range of 0-100 mu M and Necrostatin-1 with the effective concentration range of 0-40 mu M, and is divided into the following groups;
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 rest components are as follows: ADVANCED DMEM/F12 cell culture, L-WRN medium, 10% FBS, 10mM HEPS, 2mM Gluta-max, 500U/mL penicillin-streptomycin, 1 XN-2 additive, 1 XB 27 additive, 10 μ M Y27632.
The following "human colorectal cancer organoid growth medium" refers to "the remaining components" that do not contain Z-VAD-FMK and Necrostatin-1.
According to an embodiment of the invention, the final concentration of HEPES in the medium is 10mM.
According to an embodiment of the invention, the Gluta-max final concentration in the medium is 2mM.
According to an embodiment of the invention, the final concentration of penicillin in the medium is 500U/ml.
According to an embodiment of the invention, the final concentration of streptomycin in the medium is 500U/ml.
According to an embodiment of the invention, the final concentration of Y27632 in the medium is 10. Mu.M.
The term "the final concentration of the N-2 additive and the B27 additive in the medium was 1×" means that the N2 and B27 products were 100× in concentration, and the final concentrations of N2 and B27 in the medium were diluted to 1× when the medium was prepared.
Specifically, in this example, the cells are human colorectal cancer organoid cells.
The method specifically comprises the following steps:
(1) Opening a water bath kettle to regulate the temperature to 37 ℃, and dissolving matrigel on ice in advance;
(2) Taking out the frozen human colorectal cancer organoids, immersing the frozen human colorectal cancer organoids in a water bath kettle at 37 ℃ and shaking the frozen human colorectal cancer organoids from time to enable the frozen human colorectal cancer organoids to melt as soon as possible;
(3) Adding 5ml of human colorectal cancer organoid growth culture solution into a 15ml centrifuge tube, adding the thawed cell suspension into the centrifuge tube, gently mixing, and centrifuging (1500 rpm,5 min);
(4) Preheating a 96-well plate for 10min in a incubator; discarding the supernatant fluid after the centrifugation in the step (3) to obtain a cell suspension, fully mixing the cell suspension and Matrigel glue according to the volume ratio of 1:9 to avoid bubble generation, dripping 5 μl of the mixture into a 96-well plate, and placing the 96-well plate into an incubator for about 15min to solidify the Matrigel glue;
(5) Taking out the 96-well plate, adding the human colorectal cancer organoid resuscitation solution prepared according to the group, and placing the solution into an incubator for continuous culture at 37 ℃ and 5 vol percent of CO 2;
(6) After the organoid resuscitation fluid is cultured for different time, the organoid growth culture fluid is changed into the anthropogenic colorectal cancer organoid growth culture fluid for continuous culture, and the organoid growth condition is monitored and observed periodically during the resuscitation period and the image is acquired; all images herein were taken on day 10 after organoid resuscitation and the results are shown;
(7) Using ImageJ software to count the number and size of organoids;
(8) The results were calculated and plotted using GraphPad software.
As can be seen from FIGS. 1,2 and 3, when human colorectal cancer organoids are recovered, Z-VAD-FMK+Necrostatin-1 with different concentrations is added into the human colorectal cancer organoid growth culture solution, and in the range of Z-VAD-FMK (0-25 mu M) +Necrostatin-1 (0-10 mu M), the greater the concentration, the strongest organoid growth capacity is achieved when the concentration is Z-VAD-FMK (25 mu M) +Necrostatin-1 (10 mu M), and the range of Z-VAD-FMK (25-100 mu M) +Necrostatin-1 (10-40 mu M) is the gentle trend of the organoid quantity and volume, so that Z-VAD-FMK (25 mu M) +Necrostatin-1 (10 mu M) is the optimal concentration of the recovery solution;
As shown in figures 4, 5 and 6, when human colorectal cancer organoids are recovered, Z-VAD-FMK (25 mu M) +Necrostatin-1 (10 mu M) is treated for different times, organoids are recovered more efficiently within 0-48h along with the increase of the action time, and organoids grow gradually within 48-168h, so that Z-VAD-FMK (25 mu M) +Necrostatin-1 (10 mu M) is treated for 48h as the optimal mode of recovery of human colorectal cancer.
In view of the above optimal drug concentration and duration of action, the inventors compared the effect of resuscitation fluid containing a combination of a single drug and a dual drug on human colorectal cancer organoids. As shown in FIG. 7, when human colorectal cancer organoids are recovered, Z-VAD-FMK (25 mu M) or Necrostatin-1 (10 mu M) is treated alone for 48 hours, and the quantity of organoids is obviously lower than that of Z-VAD-FMK (25 mu M) +Necrostatin-1 (10 mu M) for 48 hours, which shows that the two are combined in a proper proportion to obtain a synergistic technical effect.
Example 2:
This example provides a method of immunofluorescence staining of human colorectal organoids, in this example, rabbit anti-Ki 67 antibody (1:200), alexFluo594 94 goat anti-rabbit secondary antibody (1:500),
The method specifically comprises the following steps:
(1) Organoid fixation: stopping culturing organoids 15-20 days after resuscitating, discarding culture solution, washing twice with PBS, adding cell recovery solution on ice to digest and release organoids, removing recovery solution after organoids naturally subside, adding 4 vol% paraformaldehyde, and fixing for 30min;
(2) Dehydrating: resuspending the immobilized organoids with agarose (thawed by heating) at a concentration of 2vol.% and then dehydrating the organoids in a tissue dehydrator;
(3) Embedding and slicing: embedding the dehydrated organoids in a tissue embedding machine, and slicing the tissue on a slicing machine, wherein the thickness of the tissue is about 5 mu m;
(4) Baking slices: taking paraffin embedded slices until the slices are baked for about 2 hours in an oven;
(5) Paraffin-embedded sections were dewaxed and hydrated: paraffin slicing the slices were sequentially put into xylene i 15min, xylene ii 15min,100% absolute ethanol i 10min,100% absolute ethanol ii 5min,90 vol.% ethanol 5min,80 vol.% ethanol 5min, and flow water washed for 5min;
(6) Antigen retrieval: adopting EDTA antigen repairing method, heating dewaxed hydrated glass slide in (5) until repairing liquid is bubbled, and maintaining for 15min, wherein buffer liquid is prevented from evaporating excessively in the process, and the glass slide is not dried. After repair is completed, naturally cooling, and washing for 10min by running water;
(7) Sealing, namely uniformly covering tissues by using sealing liquid, and sealing for 45-60min at room temperature;
(8) Incubating primary antibodies: diluting target antibody (1:200) with sealing solution, dripping antibody to cover tissue, incubating overnight at 4deg.C in a wet box, incubating for about 24 hr, slicing, washing with PBS for 5min on a shaker for 3 times;
(9) Incubating a secondary antibody: dripping secondary antibody covering tissue of the corresponding species with the primary antibody, incubating for 40-60min at normal temperature in dark place, and cleaning with PBS for 3 times and 5min each time;
(10) Washing off unbound secondary antibody and counterstaining DAPI, sealing with anti-fluorescence quenching sealing tablet, and photographing with Zeiss three-dimensional light cutting imaging system.
As shown in FIG. 8, the Ki67 immunofluorescence results show that compared with the resuscitation fluid-free group, the resuscitation fluid Z-VAD-FMK (25 mu M) +Necrostatin-1 (10 mu M) acts for 48 hours, the proliferation capacity of the organoid is not obviously changed after the completion of the resuscitation, and the biological genetic stability of the organoid is not changed by the resuscitation fluid, so that the organoid resuscitation efficiency is improved and the original biological genetic characteristics of the organoid are not influenced.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the 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.
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