CN114854691A - Novel colorectal cancer organoid culture method - Google Patents

Novel colorectal cancer organoid culture method Download PDF

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CN114854691A
CN114854691A CN202210581495.6A CN202210581495A CN114854691A CN 114854691 A CN114854691 A CN 114854691A CN 202210581495 A CN202210581495 A CN 202210581495A CN 114854691 A CN114854691 A CN 114854691A
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stem cells
colorectal cancer
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王磊
李欣磊
耿双艳
赵艳昱
赵树凡
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Abstract

The invention discloses a novel colorectal cancer organoid culture method, which comprises the following steps: a cell treatment step: taking down a part of tumor tissues, putting a sample taken down by an operation into a tissue protection solution, transporting the sample into a laboratory super clean bench, repeatedly cleaning the cancer tissues by using a tissue cleaning solution, shearing the rest cancer tissues into 1xmm2 fragments by using sterile scissors, adding a digestive juice, digesting for 1-3 hours by using a table concentrator at 37 ℃, centrifuging, discarding the supernatant, adding physiological saline, filtering out tissue blocks by using a cell filter screen, adding a red cell lysate if more red cells are added, cracking off the red cells, performing double-antibody CD44 and EpCAM staining on the digested single cells, and performing separation culture: the separated cells are incubated by PI to eliminate dead cells, and the method is characterized in that cd44 positive cancer stem cells and cd44 negative non-stem cells are separated and cultured, and then cd44 positive stem cells are cultured after being amplified so as to improve the success rate of tumor organoid culture.

Description

Novel colorectal cancer organoid culture method
Technical Field
The invention relates to the technical field of biological medicines, in particular to a method for culturing a novel colorectal cancer organoid.
Background
Colorectal cancer organoids are articles for biomedical technology, are a powerful research tool for in vitro modeling and research of human cancers, are cultured in vitro using cancer tissues obtained in surgery, and can reproduce pathobiological key characteristics of original tissues through in vitro self-assembly three-dimensional reconstruction, and can perform functional tests and drug screening of colorectal cancer. Usually, colorectal cancer organoid culture is to directly culture tumor tissues, and the culture method is to simulate the in vivo tumor microenvironment. The current practical culture problem is that most patient samples are subjected to surgery or radiotherapy and chemotherapy, which results in the reduction of the number of tumor stem cells in tumor tissues and further results in culture failure or unexpected organ size, thus being unfavorable for drug screening experiments. Research shows that the part of the removed tumor tissue is mixed with cd44 positive cancer stem cells and cd44 negative cancer cells which are not stem cells, and the main reason for determining organoid formation is cancer stem cells, and the cd44 positive cancer stem cells and cd44 negative non-stem cells are mixed and cultured, and the cd44 positive cancer stem cells are not separated and cultured, so that few tumor stem cells exist in a sample, and organoid structures are difficult to culture. Therefore, there is a need to develop a new organoid culture method.
Disclosure of Invention
In order to solve the problems of the background art, the present invention provides a novel method for culturing colorectal cancer organoids, which has the advantages of separating, culturing, amplifying and redifferentiating cd 44-positive cancer stem cells, and solves the problems of low culture success rate and unexpected organoid size caused by the conventional culture method for colorectal cancer organoids.
In order to achieve the purpose, the invention provides the following technical scheme: a method for culturing a novel colorectal cancer organoid comprises the following steps:
a cell treatment step: taking off a part of tumor tissues, putting a sample taken off by an operation into a tissue protection solution, conveying the sample into a laboratory super clean bench, repeatedly cleaning the cancer tissues by using a tissue cleaning solution, shearing the rest cancer tissues into 1xmm2 fragments by using sterile scissors, adding a digestive juice, digesting for 1-3 hours by using a shaking table at 37 ℃, centrifuging, discarding the supernatant, adding a physiological saline, filtering out tissue blocks by using a cell filter screen, adding a red cell lysate if more red cells are added, cracking the red cells, and dyeing the digested single cells by using double-antibody CD44 and EpCAM;
a separation culture step: isolated cells were incubated with PI to exclude dead cells, background staining levels were determined using appropriate isotype-matched control monoclonal antibodies, cell staining to exclude hematopoietic cells using FITC-conjugated anti-EpCAM antibodies and Brilliant Violet 421-conjugated anti-CD 44 antibodies, cells were analyzed and sorted using the BD FACS Aria 3 cell sorting system, cells were sorted twice with > 95% purity and reanalyzed to avoid and minimize the effects of contamination, CD44 positive cancer stem cells and CD44 negative non-stem cells were placed in a flow cytometer, then CD44 and EpCAM double positive cancer stem cells and other non-cancer stem cells were separated by flow cytometer fluorescent identification, and the two separate different cells were cultured separately.
In the isolation and culture step, the culture medium in the device for culturing cd 44-positive cancer stem cells is preferably prepared from: icabetadine sodium with a final concentration of 1 μ g/mL; vitamin P4 at a final concentration of 100. mu.M; human recombinant protein R-Spondin1 at a final concentration of 30 ng/mL; noggin, final concentration 100 ng/mL; the final concentration of the human recombinant protein rhEGF is 8 ng/mL; 4-hydroxyethylpiperazine ethanesulfonic acid at a final concentration of 1 mM; L-alanyl-L-glutamine at a final concentration of 1 ×; double resistance to the cyan chain, 1x final concentration; additive N2, final concentration 1 ×; b27 additive, final concentration 1 ×; N-acetyl-L-cysteine at a final concentration of 0.09 mM; alk inhibitor A83-01 at a final concentration of 0.5. mu.M; 4-2-5-1H-imidazole, final concentration 0.3. mu.M; gastrin, final concentration 10 nM; a non-essential amino acid solution, final concentration 1 ×; nicotinamide, final concentration 5mM and Advance DMEM/F-12 medium to 50 mL.
In the isolation and culture step, the components of the culture solution in the device for culturing cd 44-positive cancer stem cells are preferably as follows: icabetadine sodium with a final concentration of 50 μ g/mL; vitamin P4 at a final concentration of 50 μ M; human recombinant protein R-Spondin1 at a final concentration of 40 ng/mL; noggin, final concentration 90 ng/mL; the final concentration of the human recombinant protein rhEGF is 9 ng/mL; 4-hydroxyethylpiperazine ethanesulfonic acid at a final concentration of 1 mM; L-alanyl-L-glutamine at a final concentration of 1 ×; double resistance to the cyan chain, 1x final concentration; additive N2, final concentration 1 ×; b27 additive, final concentration 1 ×; N-acetyl-L-cysteine at a final concentration of 0.11 mM; alk inhibitor A83-01 at a final concentration of 0.4. mu.M; 4-2-5-1H-imidazole, final concentration 0.4. mu.M; gastrin, final concentration 10 nM; a non-essential amino acid solution, final concentration 1 ×; nicotinamide, final concentration 5mM and Advance DMEM/F-12 medium to 50 mL.
In the isolation and culture step, the components of the culture solution in the device for culturing cd 44-positive cancer stem cells are preferably as follows: icabetadine sodium with a final concentration of 100 mug/mL; vitamin P4 at a final concentration of 1 μ M; human recombinant protein R-Spondin1 at a final concentration of 50 ng/mL; noggin, final concentration 80 ng/mL; the final concentration of the human recombinant protein rhEGF is 10 ng/mL; 4-hydroxyethylpiperazine ethanesulfonic acid at a final concentration of 1 mM; L-alanyl-L-glutamine at a final concentration of 1 ×; double resistance to the cyan chain, 1x final concentration; additive N2, final concentration 1 ×; b27 additive, final concentration 1 ×; N-acetyl-L-cysteine at a final concentration of 0.12 mM; alk inhibitor A83-01 at a final concentration of 0.3. mu.M; 4-2-5-1H-imidazole, final concentration 0.5. mu.M; gastrin, final concentration 10 nM; a non-essential amino acid solution, final concentration 1 ×; nicotinamide, final concentration 5mM and Advance DMEM/F-12 medium to 50 mL.
Preferably, in the isolation culture step, the culture medium components: icatsup sodium, the final concentration is 1 mug/mL-100 mug/mL; vitamin P4, with a final concentration of 1-100 μ M; the final concentration of the human recombinant protein R-Spondin1 is 30ng/mL-50 ng/mL; noggin, final concentration 80ng/mL-100 ng/mL; the final concentration of the human recombinant protein rhEGF is 8ng/mL-10 ng/mL; 4-hydroxyethylpiperazine ethanesulfonic acid, final concentration 1 mM; L-alanyl-L-glutamine, final concentration 0.9 ×; double resistance of cyan chain, final concentration 0.9 ×; n2 additive, final concentration 0.9 ×; b27 additive, final concentration 0.9 ×; N-acetyl-L-cysteine at a final concentration of 0.09mM to 0.124 mM; alk inhibitor A83-01, with a final concentration of 0.3-0.5. mu.M; 4-2-5-1H-imidazole, final concentration 0.3-0.5. mu.M; gastrin, final concentration 9 nM; a non-essential amino acid solution, final concentration 0.9 ×; nicotinamide, 4mM final concentration and advanced DMEM/F-12 medium to 50mL are placed inside the mixing device, mixed uniformly by the mixing device to form a culture solution, and the culture solution is poured into the culture device from the inside of the mixing device.
Preferably, the feed port and the discharge port of the mixing device are provided with sealing plates, so that the interior of the mixing device can be protected from dust by the sealing plates, and the situation that external dust enters the interior of the mixing device to influence the preparation of the culture solution is avoided.
Preferably, the feeding port of the culture apparatus is provided with a cover plate, and the cover plate can prevent external dust from entering the culture apparatus.
Preferably, the double green chain antibody comprises 10000U/mL of penicillin and 10mg/mL of streptomycin.
Compared with the prior art, the invention has the following beneficial effects:
1. the cd44 positive cancer stem cells are placed in a culture device, the cd44 positive cancer stem cells are cultured by a culture solution in the culture device, then the cd44 negative non-stem cells are placed in another culture device, and the cd44 negative non-stem cells are cultured by the culture solution in the culture device, so that the cd44 positive cancer stem cells and the cd44 negative non-stem cells can be separated and cultured, the cd44 positive stem cells are separated by a flow cytometry technology, and the culture success rate of tumor organs can be greatly improved by culturing the cd44 positive cancer stem cells.
Drawings
FIG. 1 is a diagram of colorectal cancer organoids cultured by a conventional method;
FIG. 2 is a schematic representation of sorting using a flow cytometer;
FIG. 3 is a comparison graph of organoids cultured with CD 44-positive cancer stem cells and CD 44-negative non-cancer stem cells.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
A method for culturing a novel colorectal cancer organoid comprises the following steps:
a cell treatment step: taking off a part of tumor tissues, putting a sample taken off by an operation into a tissue protection solution, conveying the sample into a laboratory super clean bench, repeatedly cleaning the cancer tissues by using a tissue cleaning solution, shearing the rest cancer tissues into 1xmm2 fragments by using sterile scissors, adding a digestive juice, digesting for 1-3 hours by using a shaking table at 37 ℃, centrifuging, discarding the supernatant, adding a physiological saline, filtering out tissue blocks by using a cell filter screen, adding a red cell lysate if more red cells are added, cracking the red cells, and dyeing the digested single cells by using double-antibody CD44 and EpCAM;
a separation culture step: isolated cells were incubated with PI to exclude dead cells, background staining levels were determined using appropriate isotype-matched control monoclonal antibodies, cell staining to exclude hematopoietic cells using FITC-conjugated anti-EpCAM antibodies and Brilliant Violet 421-conjugated anti-CD 44 antibodies, cells were analyzed and sorted using the BD FACS Aria 3 cell sorting system, cells were sorted twice with > 95% purity and reanalyzed to avoid and minimize the effects of contamination, CD44 positive cancer stem cells and CD44 negative non-stem cells were placed in a flow cytometer, then CD44 and EpCAM double positive cancer stem cells and other non-cancer stem cells were separated by flow cytometer fluorescent identification, then two different cells were cultured separately, with a medium composition of sodium ikabytate at a final concentration of 1 μ g/mL; vitamin P4 at a final concentration of 100. mu.M; human recombinant protein R-Spondin1 at a final concentration of 30 ng/mL; noggin, final concentration 100 ng/mL; the final concentration of the human recombinant protein rhEGF is 8 ng/mL; 4-hydroxyethylpiperazine ethanesulfonic acid at a final concentration of 1 mM; L-alanyl-L-glutamine at a final concentration of 1 ×; double resistance to the cyan chain, 1x final concentration; additive N2, final concentration 1 ×; b27 additive, final concentration 1 ×; N-acetyl-L-cysteine at a final concentration of 0.09 mM; alk inhibitor A83-01 at a final concentration of 0.5. mu.M; 4-2-5-1H-imidazole, final concentration 0.3. mu.M; gastrin, final concentration 10 nM; a non-essential amino acid solution, final concentration 1 ×; nicotinamide, final concentration 5mM and Advance DMEM/F-12 medium to 50 mL.
Example two
A method for culturing a novel colorectal cancer organoid comprises the following steps:
a cell treatment step: taking off a part of tumor tissues, putting a sample taken off by an operation into a tissue protection solution, conveying the sample into a laboratory super clean bench, repeatedly cleaning the cancer tissues by using a tissue cleaning solution, shearing the rest cancer tissues into 1xmm2 fragments by using sterile scissors, adding a digestive juice, digesting for 1-3 hours by using a shaking table at 37 ℃, centrifuging, discarding the supernatant, adding a physiological saline, filtering out tissue blocks by using a cell filter screen, adding a red cell lysate if more red cells are added, cracking the red cells, and dyeing the digested single cells by using double-antibody CD44 and EpCAM;
a separation culture step: isolated cells were incubated with PI to exclude dead cells, background staining was determined using appropriate isotype-matched control monoclonal antibodies, cell staining was to exclude hematopoietic cells using FITC-conjugated anti-EpCAM antibodies and Brilliant Violet 421-conjugated anti-CD 44 antibodies, cells were analyzed and sorted using the BD FACS Aria 3 cell sorting system, cells were sorted twice with a purity of > 95% and re-analyzed to avoid and minimize the effects of contamination, CD44 positive cancer stem cells and CD44 negative non-stem cells were placed in a flow cytometer, then CD44 and EpCAM double positive cancer stem cells and other non-cancer stem cells were separated by flow cytometer fluorescence identification, then the two different cells isolated were cultured separately, CD44 positive cancer stem cells were cultured by culture fluid inside the culture device, the culture fluid composition was Icajadit sodium, the final concentration is 50 mug/mL; vitamin P4 at a final concentration of 50 μ M; human recombinant protein R-Spondin1 at a final concentration of 40 ng/mL; noggin, final concentration 90 ng/mL; human recombinant protein rhEGF with final concentration of 9 ng/mL; 4-hydroxyethylpiperazine ethanesulfonic acid at a final concentration of 1 mM; L-alanyl-L-glutamine at a final concentration of 1 ×; double resistance to the cyan chain, 1x final concentration; additive N2, final concentration 1 ×; b27 additive, final concentration 1 ×; N-acetyl-L-cysteine at a final concentration of 0.11 mM; alk inhibitor A83-01 at a final concentration of 0.4. mu.M; 4-2-5-1H-imidazole, final concentration 0.4. mu.M; gastrin, final concentration 10 nM; a non-essential amino acid solution, final concentration 1 ×; nicotinamide, final concentration 5mM and Advance DMEM/F-12 medium to 50 mL.
EXAMPLE III
A method for culturing a novel colorectal cancer organoid comprises the following steps:
a cell treatment step: taking off a part of tumor tissues, putting a sample taken off by an operation into a tissue protection solution, conveying the sample into a laboratory super clean bench, repeatedly cleaning the cancer tissues by using a tissue cleaning solution, shearing the rest cancer tissues into 1xmm2 fragments by using sterile scissors, adding a digestive juice, digesting for 1-3 hours by using a shaking table at 37 ℃, centrifuging, discarding the supernatant, adding a physiological saline, filtering out tissue blocks by using a cell filter screen, adding a red cell lysate if more red cells are added, cracking the red cells, and dyeing the digested single cells by using double-antibody CD44 and EpCAM;
a separation culture step: isolated cells were incubated with PI to exclude dead cells, background staining was determined using appropriate isotype-matched control monoclonal antibodies, cell staining was to exclude hematopoietic cells using FITC-conjugated anti-EpCAM antibodies and Brilliant Violet 421-conjugated anti-CD 44 antibodies, cells were analyzed and sorted using the BD FACS Aria 3 cell sorting system, cells were sorted twice with a purity of > 95% and re-analyzed to avoid and minimize the effects of contamination, CD44 positive cancer stem cells and CD44 negative non-stem cells were placed in a flow cytometer, then CD44 and EpCAM double positive cancer stem cells and other non-cancer stem cells were separated by flow cytometer fluorescence identification, then the two different cells isolated were cultured separately, CD44 positive cancer stem cells were cultured by culture fluid inside the culture device, the culture fluid composition was Icajadit sodium, the final concentration is 100 mug/mL; vitamin P4 at a final concentration of 1 μ M; human recombinant protein R-Spondin1 at a final concentration of 50 ng/mL; noggin, final concentration 80 ng/mL; the final concentration of the human recombinant protein rhEGF is 10 ng/mL; 4-hydroxyethylpiperazine ethanesulfonic acid at a final concentration of 1 mM; L-alanyl-L-glutamine at a final concentration of 1 ×; double resistance to the cyan chain, 1x final concentration; additive N2, final concentration 1 ×; b27 additive, final concentration 1 ×; N-acetyl-L-cysteine at a final concentration of 0.12 mM; alk inhibitor A83-01 at a final concentration of 0.3. mu.M; 4-2-5-1H-imidazole, final concentration 0.5. mu.M; gastrin, final concentration 10 nM; a non-essential amino acid solution, final concentration 1 ×; nicotinamide, final concentration 5mM and Advance DMEM/F-12 medium to 50 mL.
From FIG. 3, it can be seen that the number of tumor organoids cultured with CD 44-positive cancer stem cells is increased compared to the number of tumor organoids obtained by the conventional method, and the tumor organoids can hardly be cultured with CD 44-negative non-cancer stem cells.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (8)

1. A novel colorectal cancer organoid culture method is characterized in that: the method comprises the following steps:
a cell treatment step: taking off a part of tumor tissues of a patient, putting a sample taken off by an operation into a tissue protection solution, transporting the sample into a laboratory super clean bench, repeatedly cleaning the cancer tissues by using a tissue cleaning solution, shearing the rest cancer tissues into 2 fragments with the size of 1xmm by using sterile scissors, adding a digestive juice, digesting for 1-3 hours by using a shaking table at 37 ℃, centrifuging, removing the supernatant, adding physiological saline, filtering out tissue blocks by using a cell filter screen, adding a red cell lysate if more red cells are added, cracking the red cells, and dyeing the digested single cells by using double-antibody CD44 and EpCAM;
a separation culture step: isolated cells were incubated with PI to exclude dead cells, background staining levels were determined using appropriate isotype-matched control monoclonal antibodies, cell staining to exclude hematopoietic cells using FITC-conjugated anti-EpCAM antibodies and Brilliant Violet 421-conjugated anti-CD 44 antibodies, cells were analyzed and sorted using the BD FACS Aria 3 cell sorting system, cells were sorted twice with > 95% purity and reanalyzed to avoid and minimize the effects of contamination, CD44 positive cancer stem cells and CD44 negative non-stem cells were placed in a flow cytometer, then CD44 and EpCAM double positive cancer stem cells and other non-cancer stem cells were separated by flow cytometer fluorescent identification, and the two separate different cells were cultured separately.
2. The method of claim 1, wherein the culture of a novel colorectal cancer organoid comprises: the separation culture step comprises the following steps of culturing cd44 positive cancer stem cells by using the culture solution in the device as raw materials: icabetadine sodium with a final concentration of 1 μ g/mL; vitamin P4 at a final concentration of 100. mu.M; human recombinant protein R-Spondin1 at a final concentration of 30 ng/mL; noggin, final concentration 100 ng/mL; the final concentration of the human recombinant protein rhEGF is 8 ng/mL; 4-hydroxyethyl piperazine ethanesulfonic acid at a final concentration of 1 mM; L-alanyl-L-glutamine at a final concentration of 1 ×; double resistance to the cyan chain, 1x final concentration; additive N2, final concentration 1 ×; b27 additive, final concentration 1 ×; N-acetyl-L-cysteine at a final concentration of 0.09 mM; alk inhibitor A83-01 at a final concentration of 0.5. mu.M; 4-2-5-1H-imidazole, final concentration 0.3. mu.M; gastrin, final concentration 10 nM; a non-essential amino acid solution, final concentration 1 ×; nicotinamide, final concentration 5mM and Advance DMEM/F-12 medium to 50 mL.
3. The method of claim 2, wherein the culture of a novel colorectal cancer organoid comprises: the separation and culture step is carried out in such a manner that the components of a culture solution in the device for culturing cd 44-positive cancer stem cells are: icabetadine sodium with a final concentration of 50 μ g/mL; vitamin P4 at a final concentration of 50 μ M; human recombinant protein R-Spondin1 at a final concentration of 40 ng/mL; noggin, final concentration 90 ng/mL; the final concentration of the human recombinant protein rhEGF is 9 ng/mL; 4-hydroxyethylpiperazine ethanesulfonic acid at a final concentration of 1 mM; L-alanyl-L-glutamine at a final concentration of 1 ×; double resistance to the cyan chain, 1x final concentration; additive N2, final concentration 1 ×; b27 additive, final concentration 1 ×; N-acetyl-L-cysteine at a final concentration of 0.11 mM; alk inhibitor A83-01 at a final concentration of 0.4. mu.M; 4-2-5-1H-imidazole, final concentration 0.4. mu.M; gastrin, final concentration 10 nM; a non-essential amino acid solution, final concentration 1 ×; nicotinamide, final concentration 5mM and Advance DMEM/F-12 medium to 50 mL.
4. The method of claim 3, wherein the culture of a novel colorectal cancer organoid comprises: the separation and culture step is carried out in such a manner that the components of a culture solution in the device for culturing cd 44-positive cancer stem cells are: incarbate sodium with a final concentration of 100 mug/mL; vitamin P4 at a final concentration of 1 μ M; human recombinant protein R-Spondin1 at a final concentration of 50 ng/mL; noggin, final concentration 80 ng/mL; the final concentration of the human recombinant protein rhEGF is 10 ng/mL; 4-hydroxyethylpiperazine ethanesulfonic acid at a final concentration of 1 mM; L-alanyl-L-glutamine at 1x final concentration; double resistance to the cyan chain, 1x final concentration; additive N2, final concentration 1 ×; b27 additive, final concentration 1 ×; N-acetyl-L-cysteine at a final concentration of 0.12 mM; alk inhibitor A83-01 at a final concentration of 0.3. mu.M; 4-2-5-1H-imidazole, final concentration 0.5. mu.M; gastrin, final concentration 10 nM; a non-essential amino acid solution, final concentration 1 ×; nicotinamide, final concentration 5mM and Advance DMEM/F-12 medium to 50 mL.
5. The method of claim 4, wherein the culture of the colorectal cancer organoid comprises: the separation culture step comprises the following steps of: icatsup sodium, the final concentration is 1 mug/mL-100 mug/mL; vitamin P4, with a final concentration of 1-100 μ M; the final concentration of the human recombinant protein R-Spondin1 is 30ng/mL-50 ng/mL; noggin, final concentration 80ng/mL-100 ng/mL; the final concentration of the human recombinant protein rhEGF is 8ng/mL-10 ng/mL; 4-hydroxyethylpiperazine ethanesulfonic acid, final concentration 1 mM; L-alanyl-L-glutamine, final concentration 0.9 ×; double resistance of cyan chain, final concentration 0.9 ×; n2 additive, final concentration 0.9 ×; b27 additive, final concentration 0.9 ×; N-acetyl-L-cysteine at a final concentration of 0.09mM to 0.124 mM; alk inhibitor A83-01, with a final concentration of 0.3-0.5. mu.M; 4-2-5-1H-imidazole, final concentration 0.3-0.5. mu.M; gastrin, final concentration 9 nM; a non-essential amino acid solution, final concentration 0.9 ×; nicotinamide, 4mM final concentration and advanced DMEM/F-12 medium to 50mL are placed inside the mixing device, mixed uniformly by the mixing device to form a culture solution, and the culture solution is poured into the culture device from the inside of the mixing device.
6. The method of claim 5, wherein the culture of the colorectal cancer organoid comprises: the feed inlet and the discharge gate of mixing arrangement all are provided with the closing plate, through setting up the closing plate, can prevent dust mixing arrangement's inside, avoid outside dust to get into mixing arrangement's inside to influence the preparation of culture solution.
7. The method of claim 6, wherein the culture of a novel colorectal cancer organoid comprises: the feed inlet of culture apparatus is provided with the apron, through setting up the apron, can prevent that outside dust from getting into culture apparatus's inside.
8. The method of claim 7, wherein the culture of the colorectal cancer organoid comprises: the double-antibody of the cyan chain contains 10000U/mL penicillin and 10mg/mL streptomycin.
CN202210581495.6A 2022-05-26 2022-05-26 Novel colorectal cancer organoid culture method Pending CN114854691A (en)

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