CN112813028B - Culture solution for colorectal cancer organoid culture and preparation method thereof - Google Patents

Culture solution for colorectal cancer organoid culture and preparation method thereof Download PDF

Info

Publication number
CN112813028B
CN112813028B CN202110109040.XA CN202110109040A CN112813028B CN 112813028 B CN112813028 B CN 112813028B CN 202110109040 A CN202110109040 A CN 202110109040A CN 112813028 B CN112813028 B CN 112813028B
Authority
CN
China
Prior art keywords
final concentration
culture
colorectal cancer
culture solution
solution
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202110109040.XA
Other languages
Chinese (zh)
Other versions
CN112813028A (en
Inventor
游明亮
王磊
申重阳
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hangzhou Aiming Medical Technology Co ltd
Original Assignee
Hangzhou Aiming Medical Technology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hangzhou Aiming Medical Technology Co ltd filed Critical Hangzhou Aiming Medical Technology Co ltd
Priority to CN202110109040.XA priority Critical patent/CN112813028B/en
Publication of CN112813028A publication Critical patent/CN112813028A/en
Application granted granted Critical
Publication of CN112813028B publication Critical patent/CN112813028B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • C12N2500/33—Amino acids other than alpha-amino carboxylic acids, e.g. beta-amino acids, taurine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Oncology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a culture solution for colorectal cancer organoid culture and a preparation method thereof, belonging to the technical field of biological medicine. The culture solution is prepared from the following raw materials: icatide sodium, vitamin P4, human recombinant protein R-Spond i N1, noggin, human recombinant protein rhEGF, 4-hydroxyethylpiperazineethanesulfonic acid, L-alanyl-L-glutamine, azulene bis-antibody, N2 supplement, B27 supplement, N-acetyl-L-cysteine, Al k inhibitor A83-01, 4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole, gastrin, non-essential amino acid solution, nicotinamide and Advanced DMEM/F-12 medium. The culture solution can ensure that colorectal cancer tissue cells can keep very high cell and tissue activity in the culture process, and the inherent pathological characteristics of the colorectal cancer tissue cells can be better represented.

Description

Culture solution for colorectal cancer organoid culture and preparation method thereof
Technical Field
The invention relates to a culture solution for colorectal cancer organoid culture and a preparation method thereof, belonging to the technical field of biological medicine.
Background
Colorectal cancer is one of the most common malignancies that severely threaten human health. The incidence of colorectal cancer in our country is the third in all malignant tumors. While the mortality rate of colorectal cancer accounts for the fifth of all malignancies. With the development of economy, improvement of living standard and change of life style, the incidence rate of colorectal cancer is on an increasing trend.
Colorectal cancer is a complex disease, the occurrence and development of the colorectal cancer are dynamic processes, a plurality of signal molecules are involved in interaction, a complex molecular regulation network is formed, and meanwhile, the colorectal cancer is influenced by external environmental factors, and the etiology and the occurrence and development processes of the colorectal cancer have strong individual differences and cannot be determined in a whole.
The basic mechanisms underlying the pathobiology of colorectal cancer are currently poorly understood, and the efficiency of treatment and patient survival are not significantly improved, mainly due to the lack of suitable research models. Organoid technology is a powerful tool for in vitro modeling and research of human cancers. It uses the cancer tissue obtained in surgery to culture in vitro, and through in vitro self-assembly three-dimensional (3D) reconstruction, the obtained organoids can reproduce the pathobiological key characteristics of the original tissue, and can be used for functional testing and drug screening of colorectal cancer. Patient-derived cancer tissue organoids are advanced preclinical tools for studying the pathobiological properties of colorectal cancer and for (personalized) drug screening.
Studies have shown that tumor organoids themselves grow at a slower rate than their corresponding normal tissue organoids, and even in many cases at a slower rate, which may be associated with failed mitosis and cell death following division. Therefore, there is a need to avoid excessive growth of epithelial organoids of surviving normal tissue in tumor organoids during tumor organoid culture, and tumor organoids must be cultured using pure tumor cell samples or under selective culture conditions.
In view of the above, there is a need to develop a culture solution for colorectal cancer organoid culture and a preparation method thereof, so as to solve the deficiencies of the prior art.
Disclosure of Invention
One of the purposes of the invention is to provide a culture solution for colorectal cancer organoid culture. The culture solution of the invention contains a plurality of cytokines, signal path regulating factors, amino acids, serum-free additives and the like which are required by the culture of the human colorectal cancer tissue cells, and the various cytokines and regulating factors are directly and closely influenced and coordinately matched with each other, so that the colorectal cancer tissue cells can keep very high cell and tissue activity in the culture process, the inherent pathological characteristics of the colorectal cancer tissue cells can be better represented, and the colorectal cancer tissue cells are highly consistent with the pathobiological characteristics of original tissues and the high-frequency mutation genes of rectal cancer tumors.
The technical scheme for solving the technical problems is as follows: a culture solution for colorectal cancer organoid culture, wherein each 50mL of the culture solution is prepared from the following raw materials: icatsup sodium with a final concentration of 1-100 μ g/mL; vitamin P4 at a final concentration of 1-100. mu.M; the final concentration of the human recombinant protein R-Spondin1 is 30ng/mL-50 ng/mL; noggin with final concentration of 80-100 ng/mL; the final concentration of the human recombinant protein rhEGF is 8ng/mL-10 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 of green chain, and the final concentration is 1X; additive N2, final concentration 1 ×; b27 additive, final concentration 1 ×; N-acetyl-L-cysteine at a final concentration of 0.09mM-0.125 mM; alk inhibitor A83-01 with final concentration of 0.3-0.5. mu.M; 4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole at a final concentration of 0.3 μ M to 0.5 μ M; gastrin, final concentration 10 nM; a non-essential amino acid solution at a final concentration of 1 ×; nicotinamide, final concentration 5mM and Advanced DMEM/F-12 medium to 50 mL.
The principle of the culture solution for colorectal cancer organoid culture is as follows:
the oxygen concentration outside the colorectal cancer organoid cell mass is 6%, while the oxygen concentration inside the colorectal cancer organoid cell mass is only 0.5%, and hypoxia causes a large amount of apoptosis, affecting the developmental morphology and tissue specificity of the organoids. According to the invention, the Ecabet sodium (English name is Ecabet sodium) is added into the culture solution, so that the apoptosis of cells in the organoid can be reduced, the activity of an inner cell mass can be improved, the organoid can be prevented from hollowing by inhibiting the generation of ROS active oxygen, and the integrity of the organoid can be further maintained.
The colorectal cancer organoid cells contain a large amount of mutant genes, a large amount of proteins with abnormal structures can be generated, the abnormal proteins cannot be normally folded and glycosylated, and are accumulated in endoplasmic reticulum in a large amount, so that the stress response of activating the endoplasmic reticulum is caused, the apoptosis of the cells is caused in a large amount, the growth and the survival of the colorectal cancer organoid are influenced, and the display of the colorectal cancer tumor high-frequency mutant genes in the organoid is also influenced. According to the invention, vitamin P4 (Traerotin) is added into the culture solution, and the apoptosis can be further reduced by inhibiting an endoplasmic reticulum stress mediated apoptosis pathway, so that the pathological characteristics of the high-frequency mutant gene of the rectal cancer tumor can be more truly displayed.
In addition, the culture solution also comprises the following raw materials:
1. human recombinant protein R-Spondin1, as a ligand of LGR5, activates a classical Wnt signaling pathway after being combined with LGR5 to promote invasive metastatic capacity of colorectal cancer. The human recombinant protein R-Spondin1 is commercially available, for example, from R & D Systems, Inc. under the reference 4645-RS-100.
2. Noggin, also known as Noggin, is an inhibitor of BMP and is capable of antagonizing BMP and promoting crypt proliferation. The BMP (Bone Morphogenetic Protein) signal channel is used as the Wnt signal channel negative regulation signal. The noggin is commercially available, for example, from R & D Systems under the trade designation 6057-NG-025.
3. The human recombinant protein rhEGF is used as mitogen, is highly expressed in crypts of small intestine, and has important effect on proliferation of stem cells and TA cells. The recombinant human rhEGF is commercially available, for example, from Sigma under the reference number E9644.
4. 4-hydroxyethyl piperazine ethanesulfonic acid, which is HEPES under the English name, has no toxic effect on cells, is a hydrogen ion buffer, and can control a constant pH range for a long time. The above 4-hydroxyethylpiperazine ethanesulfonic acid is commercially available, for example, from Gibco under the designation 15630080.
5. L-alanyl-L-glutamine, known by the English name Gluta MAX, contains dipeptides in the form of stabilized L-glutamine, L-alanyl-L-glutamine, is more stable in aqueous solution, does not degrade spontaneously, and can prevent degradation of glutamine and accumulation of ammonia during long-term culture. The L-alanyl-L-glutamine can be purchased commercially, for example, from Gibco under the trade designation 35050 × -061, 100 ×.
6. The double-resistance of the green chain has the functions of inhibiting the growth of bacteria and avoiding cell pollution. The above-mentioned cyan chain diabody is commercially available, for example, from Gibco corporation under the reference 15140122.
7. The N2 additive is a serum-free additive, is used for the mitotic anaphase of cells, can maintain the survival and growth of the cells, and does not need to add serum into a culture medium.
The N2 additive (100X) contained human holotransferrin at a final concentration of 1mM, recombinant insulin holochain at 500mg/mL, progesterone at 0.63mg/L, putrescine at 10mM and selenite at 0.52 mg/L. The N2 additive is commercially available, for example, from Thermo corporation under the accession number 17502001.
8. The B27 additive is a serum-free additive, can maintain the survival and growth of cells, and does not need to add serum into a culture medium.
The additive B27 contains biotin, DL-alpha-tocopherol acetate, DL-alpha-tocopherol, BSA, catalase, human recombinant insulin, human transferrin, superoxide dismutase, corticosterone, D-galactose, ethanolamine hydrochloric acid, reduced glutathione, L-carnitine hydrochloric acid, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite and triiodothyronine. The B27 additive is commercially available, for example, from Thermo corporation under the accession number 12587010.
9. N-acetyl-L-cysteine, known under the English name of N-acetyl-L-cysteine, can prevent DNA fragmentation of apoptosis and maintain long-term survival without other nutritional support.
The N-acetyl-L-cysteine is commercially available, for example, from Sigma under the trade name A9165.
10. The Alk inhibitor A8301 is an inhibitor of TGF-beta type I receptors ALK5, ALK4 and ALK7, and can inhibit transcription induced by ALK5, ALK4 and ALK 7. The Alk inhibitor A8301 is commercially available, for example, from Sigma under the accession number SmL 0788.
11. 4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole, having the english name SB202190, is a highly selective, potent and cell permeable inhibitor of p38 MAP kinase, binds within the ATP pocket of the active kinase and selectively inhibits the p38 α and β subtypes. The above 4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole is commercially available, for example, from Sigma under the reference number S7067.
12. Gastrin, also known by the english name Gastrin I human, is an endogenous peptide produced in the stomach that increases the secretion of gastric acid through the CCK2 receptor. The gastrin is commercially available, for example, from Sigma under the reference number G9145.
13. The Non-Essential Amino acid solution is called Non-Essential Amino Acids in English, is called NEAA in English for short, comprises 7 Non-Essential Amino Acids of L-alanine, L-glutamic acid, L-asparagine (aspartic acid), L-aspartic acid, L-proline, L-serine and glycine, can effectively improve the proportion of a cell culture medium, reduces the side effect of self production of the Non-Essential Amino Acids by cells during cell culture, promotes cell proliferation and metabolism, and is one of additives commonly used in cell culture. The above-mentioned non-essential amino acid solution can be purchased commercially, for example, from Gibco having a commercial product number of 11140-050.
14. Nicotinamide, known in English under the name Nicotinamide. In cells, nicotinic acid is used to synthesize Nicotinamide Adenine Dinucleotide (NAD) and Nicotinamide Adenine Dinucleotide Phosphate (NADP). The nicotinamide conversion pathway is very similar to that of nicotinic acid. NAD and NADP are coenzymes for a variety of enzymatic redox reactions.
The nicotinamide mentioned above is commercially available, e.g. from Sigma under the designation N0636.
15. The Advanced DMEM/F-12 medium is a mixture of a DMEM medium and an F-12 medium according to the volume ratio of 1:1, contains high-concentration nutrient substances and is favorable for cell division and proliferation.
The above-mentioned Advanced DMEM/F-12 medium is commercially available, for example, from Gibco under the reference 12634010.
In summary, the culture solution of the invention contains a plurality of cytokines, signal pathway regulatory factors, amino acids, serum-free additives and the like which are needed by the culture of the human colorectal cancer tissue cells, and the various cytokines and regulatory factors are directly and closely influenced and coordinately matched with each other, so that the colorectal cancer tissue cells can keep very high cell and tissue activity in the culture process, better represent the inherent pathological characteristics of the colorectal cancer tissue cells, and keep high consistency with the pathological biological characteristics of the original tissues and the high-frequency mutation genes of the rectal cancer tumors.
The culture solution for colorectal cancer organoid culture has the beneficial effects that:
1. the culture solution comprises a plurality of cytokines, signal channel regulation factors, amino acids, serum-free additives and the like which are required by the culture of the human colorectal cancer tissue cells, and the various cytokines and the regulation factors are directly and closely influenced and coordinately matched with each other, so that the colorectal cancer tissue cells can better express the inherent activity characteristics in the culture process, and the culture solution is highly consistent with the pathobiology characteristics of original tissues and the high-frequency mutation genes of rectal cancer tumors.
2. The invention has wide raw material source, low cost and wide application prospect.
On the basis of the technical scheme, the invention can be improved as follows.
Further, each 50mL of the culture solution is prepared from the following raw materials: icabetadine sodium with a final concentration of 1 μ g/mL; vitamin P4, final concentration 100 μ M; the final concentration of the human recombinant protein R-Spondin1 is 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 of green chain, and the final concentration is 1X; 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- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole at a final concentration of 0.3 μ M; gastrin, final concentration 10 nM; a non-essential amino acid solution at a final concentration of 1 ×; nicotinamide, final concentration 5mM and Advanced DMEM/F-12 medium to 50 mL.
The adoption of the further beneficial effects is as follows: the above is the optimum parameter, and the obtained culture solution has the best effect.
Further, each 50mL of the culture solution is prepared from the following raw materials: icabetadine sodium with a final concentration of 50 μ g/mL; vitamin P4 at a final concentration of 50 μ M; the final concentration of the human recombinant protein R-Spondin1 is 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 1 x final concentration; double resistance to the cyan chain, 1 x final concentration; n2 additive, 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- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole at a final concentration of 0.4 μ M; gastrin, final concentration 10 nM; a non-essential amino acid solution, final concentration 1 ×; nicotinamide, final concentration 5mM and Advanced DMEM/F-12 medium to 50 mL.
The adoption of the further beneficial effects is as follows: the above is the optimum parameter, and the obtained culture solution has the best effect.
Further, each 50mL of the culture solution is prepared from the following raw materials: icabetadine sodium with a final concentration of 100 mug/mL; vitamin P4, at a final concentration of 1 μ M; the final concentration of the human recombinant protein R-Spondin1 is 50 ng/mL; noggin, final concentration 80 ng/mL; human recombinant protein rhEGF with final concentration of 10 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, 1 x final concentration; additive N2, final concentration 1 ×; b27 additive, final concentration 1 ×; N-acetyl-L-cysteine at a final concentration of 0.125 mM; alk inhibitor A83-01 at a final concentration of 0.3. mu.M; 4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole at a final concentration of 0.5 μ M; gastrin, final concentration 10 nM; a non-essential amino acid solution at a final concentration of 1 ×; nicotinamide, final concentration 5mM and Advanced DMEM/F-12 medium to 50 mL.
The adoption of the further beneficial effects is as follows: the above is the optimum parameter, and the obtained culture solution has the best effect.
Further, the double green chain antibody contains 10000U/mL penicillin and 10mg/mL streptomycin.
The adoption of the method has the further beneficial effects that: penicillin and streptomycin act on gram-positive and gram-negative bacteria, respectively, and their use in combination can prevent bacterial contamination of cell cultures. Penicillin is initially purified from penicillium fungi and further alters the cell wall by directly interfering with the turnover of the bacterial cell wall, indirectly triggering the release of enzymes. Streptomycin was originally purified from streptomyces. Streptomycin inhibits protein synthesis and causes bacterial death by binding to the bacterial ribosomal 30S subunit.
The second purpose of the invention is to provide a preparation method of the culture solution for colorectal cancer organoid culture. The preparation method is simple, easy to operate, low in cost and suitable for large-scale popularization and application.
The technical scheme for solving the technical problems is as follows: a preparation method of a culture solution for colorectal cancer organoid culture comprises the following steps:
the following raw materials are respectively weighed: icatsup sodium with a final concentration of 1-100 μ g/mL; vitamin P4 at a final concentration of 1-100. mu.M; the final concentration of the human recombinant protein R-Spondin1 is 30ng/mL-50 ng/mL; noggin with final concentration of 80-100 ng/mL; the final concentration of the human recombinant protein rhEGF is 8ng/mL-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, 1 x final concentration; n2 additive, final concentration 1 ×; b27 additive, final concentration 1 ×; N-acetyl-L-cysteine at a final concentration of 0.09mM to 0.125 mM; alk inhibitor A83-01 with final concentration of 0.3-0.5 μ M; 4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole at a final concentration of 0.3 μ M to 0.5 μ M; gastrin, final concentration 10 nM; a non-essential amino acid solution at a final concentration of 1 ×; nicotinamide with the final concentration of 5mM and the Advanced DMEM/F-12 culture medium being supplemented to 50mL, and the culture solution for culturing the colorectal cancer organoids is obtained after the raw materials are uniformly mixed.
The preparation method of the culture solution for colorectal cancer organoid culture has the beneficial effects that:
the preparation method is simple, easy to operate, low in cost and suitable for large-scale popularization and application.
Drawings
FIG. 1 shows a single cell of colorectal cancer in Experimental example 5 of the present invention, with a scale of 200 μm and a magnification of 100 times.
FIG. 2 is a graph showing organoids obtained after primary culture of colorectal cancer tissue in Experimental example 5 of the present invention, with a scale of 200 μm and a magnification of 100 times.
FIG. 3 is a photograph of the follow-up 0d of continuous culture of cultured primary tumor cells in Experimental example 5 of the present invention using the culture solution of example 2, with a scale of 200 μm and a magnification of 100.
FIG. 4 is a photograph of the follow-up 3d of continuous culture of cultured primary tumor cells in Experimental example 5 of the present invention using the culture solution of example 2, with a scale of 200 μm and a magnification of 100.
FIG. 5 is a photograph of the 4 th day of continuous culture trace of primary tumor cells cultured in the culture medium of example 2 in Experimental example 5 of the present invention, with a scale of 200 μm and a magnification of 100 times.
FIG. 6 is a photograph of the follow-up 5d of continuous culture of cultured primary tumor cells in Experimental example 5 of the present invention using the culture solution of example 2, with a scale of 200 μm and a magnification of 100.
FIG. 7 is a photograph showing the following 7d of the continuous culture of primary tumor cells cultured in the culture medium of example 2 in Experimental example 5 of the present invention, with a scale of 200 μm and a magnification of 100 times
FIG. 8 is a photograph of the follow-up 0d of continuous culture of cultured primary tumor cells in Experimental example 6 of the present invention using the culture solution of comparative example 1, with a scale of 200 μm and a magnification of 100 times.
FIG. 9 is a photograph of the follow-up 3d of continuous culture of cultured primary tumor cells in Experimental example 6 of the present invention using the culture solution of comparative example 1, with a scale of 200 μm and a magnification of 100 times.
FIG. 10 is a photograph of a 4 d-follow-up continuous culture of primary tumor cells cultured in the culture solution of comparative example 1 in Experimental example 6 of the present invention, with a scale of 200 μm and a magnification of 100.
FIG. 11 is a photograph of the follow-up 5d of continuous culture of cultured primary tumor cells in Experimental example 6 of the present invention using the culture solution of comparative example 1, with a scale of 200 μm and a magnification of 100 times.
FIG. 12 is a photograph of the follow-up 7d of continuous culture of cultured primary tumor cells in Experimental example 6 of the present invention using the culture solution of comparative example 1, with a scale of 200 μm and a magnification of 100 times.
FIG. 13 is a photograph of the follow-up 0d of continuous culture of cultured primary tumor cells in Experimental example 7 of the present invention using the culture solution of comparative example 2, with a scale of 200 μm and a magnification of 100 times.
FIG. 14 is a photograph of the following 3d of continuous culture of cultured primary tumor cells in Experimental example 7 of the present invention using the culture solution of comparative example 2, with a scale of 200 μm and a magnification of 100 times.
FIG. 15 is a photograph of the 4 th day of continuous culture follow-up of primary tumor cells cultured in the culture solution of comparative example 2 in Experimental example 7 of the present invention, with a scale of 200 μm and a magnification of 100 times.
FIG. 16 is a photograph of the follow-up 5d of continuous culture of cultured primary tumor cells in Experimental example 7 of the present invention using the culture solution of comparative example 2, with a scale of 200 μm and a magnification of 100 times.
FIG. 17 is a photograph of a trace of 7d in experimental example 7 of the present invention, in which primary tumor cells were cultured using the culture solution of comparative example 2, on a scale of 200 μm at a magnification of 100.
Detailed Description
The principles and features of this invention are described below in conjunction with the following detailed drawings, which are given by way of illustration only and are not intended to limit the scope of the invention.
Example 1
The culture solution for colorectal cancer organoid culture of the present example was prepared from the following raw materials per 50 mL: incarbate sodium with a final concentration of 1 μ g/mL; vitamin P4, final concentration 100 μ M; the final concentration of the human recombinant protein R-Spondin1 is 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 1 x final concentration; double resistance to the cyan chain, 1 x final concentration; n2 additive, 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- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole at a final concentration of 0.3 μ M; gastrin, final concentration 10 nM; a non-essential amino acid solution, final concentration 1 ×; nicotinamide, final concentration 5mM and Advanced DMEM/F-12 medium to 50 mL. The double-antibody of the green chain contains 10000U/mL penicillin and 10mg/mL streptomycin.
The preparation method of the culture solution for colorectal cancer organoid culture comprises the following steps:
the following raw materials are respectively weighed: incarbate sodium with a final concentration of 1 μ g/mL; vitamin P4, final concentration 100 μ M; the final concentration of the human recombinant protein R-Spondin1 is 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 1 x final concentration; double resistance to the cyan chain, 1 x 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- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole at a final concentration of 0.3 μ M; gastrin, final concentration 10 nM; a non-essential amino acid solution at a final concentration of 1 ×; nicotinamide with the final concentration of 5mM and the Advanced DMEM/F-12 culture medium being supplemented to 50mL, and the culture solution for culturing the colorectal cancer organoids is obtained after the raw materials are uniformly mixed. After the preparation of the culture medium, the culture medium was sterilized and filtered through a 0.22 μm filter, and stored at 4 ℃ for 2 weeks.
Example 2
The culture solution for colorectal cancer organoid culture of the present example was prepared from the following raw materials per 50 mL: incarbate sodium with a final concentration of 50 μ g/mL; vitamin P4 at a final concentration of 50 μ M; the final concentration of the human recombinant protein R-Spondin1 is 40 ng/mL; noggin, final concentration 90 ng/mL; human recombinant protein rhEGF with final concentration of 9 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, 1 x 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- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole at a final concentration of 0.4 μ M; gastrin, final concentration 10 nM; a non-essential amino acid solution, final concentration 1 ×; nicotinamide, final concentration 5mM and Advanced DMEM/F-12 medium to 50 mL. The double-antibody of the green chain contains 10000U/mL penicillin and 10mg/mL streptomycin.
The preparation method of the culture solution for colorectal cancer organoid culture comprises the following steps:
the following raw materials are respectively weighed: icabetadine sodium with a final concentration of 50 μ g/mL; vitamin P4 at a final concentration of 50 μ M; the final concentration of the human recombinant protein R-Spondin1 is 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, 1 x 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- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole at a final concentration of 0.4 μ M; gastrin, final concentration 10 nM; a non-essential amino acid solution at a final concentration of 1 ×; nicotinamide with the final concentration of 5mM and the Advanced DMEM/F-12 culture medium being supplemented to 50mL, and the culture solution for culturing the colorectal cancer organoids is obtained after the raw materials are uniformly mixed. After the preparation of the culture medium, the culture medium was sterilized and filtered through a 0.22 μm filter, and stored at 4 ℃ for 2 weeks.
Example 3
The culture solution for colorectal cancer organoid culture of the present example was prepared from the following raw materials per 50 mL: incarbate sodium with a final concentration of 100 mug/mL; vitamin P4, at a final concentration of 1 μ M; the final concentration of the human recombinant protein R-Spondin1 is 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 of green chain, and the final concentration is 1X; n2 additive, final concentration 1 ×; b27 additive, final concentration 1 ×; N-acetyl-L-cysteine at a final concentration of 0.125 mM; alk inhibitor A83-01 at a final concentration of 0.3. mu.M; 4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole at a final concentration of 0.5 μ 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. The double-antibody of the green chain contains 10000U/mL penicillin and 10mg/mL streptomycin.
The preparation method of the culture solution for colorectal cancer organoid culture comprises the following steps:
the following raw materials are respectively weighed: incarbate sodium with a final concentration of 50 μ g/mL; vitamin P4, at a final concentration of 50 μ M; the final concentration of the human recombinant protein R-Spondin1 is 40 ng/mL; noggin, final concentration 90 ng/mL; the final concentration of the human recombinant protein rhEGF is 9 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 of green chain, and the final concentration is 1X; 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- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole at a final concentration of 0.4 μ M; gastrin, final concentration 10 nM; a non-essential amino acid solution, final concentration 1 ×; nicotinamide with the final concentration of 5mM and the Advanced DMEM/F-12 culture medium being supplemented to 50mL, and the culture solution for culturing the colorectal cancer organoids is obtained after the raw materials are uniformly mixed. After the preparation of the culture medium, the culture medium was sterilized and filtered through a 0.22 μm filter, and stored at 4 ℃ for 2 weeks.
Comparative example 1
The culture solution of comparative example 1 was the same as that of example 2 except that no sodium ecabet was contained. Specifically, this comparative example was made of the following raw materials per 50mL of culture solution: vitamin P4 at a final concentration of 50 μ M; the final concentration of the human recombinant protein R-Spondin1 is 40 ng/mL; noggin, final concentration 90 ng/mL; the final concentration of the human recombinant protein rhEGF is 9 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, 1 x 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- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole at a final concentration of 0.4 μ M; gastrin, final concentration 10 nM; a non-essential amino acid solution at a final concentration of 1 ×; nicotinamide, final concentration 5mM and Advanced DMEM/F-12 medium to 50 mL. The double-antibody of the green chain contains 10000U/mL penicillin and 10mg/mL streptomycin.
Comparative example 2
The culture solution of comparative example 2 was the same as that of example 2 except that vitamin P4 was not contained in the culture solution. Specifically, this comparative example was made of the following raw materials per 50mL of culture solution: incarbate sodium with a final concentration of 50 μ g/mL; the final concentration of the human recombinant protein R-Spondin1 is 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 1 x final concentration; double resistance of green chain, and the final concentration is 1X; n2 additive, 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- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole at a final concentration of 0.4 μ M; gastrin, final concentration 10 nM; a non-essential amino acid solution, final concentration 1 ×; nicotinamide, final concentration 5mM and Advanced DMEM/F-12 medium to 50 mL. The double-antibody of the green chain contains 10000U/mL penicillin and 10mg/mL streptomycin.
Experimental example 1: preparation of reagent for culturing colorectal cancer organoid
1. Specimen preservation solution
500mL of sample preservation solution is prepared from the following raw materials: fetal bovine serum, 10mL, final concentration 2%; double antibody, 5mL, final concentration of 1%; advanced DMEM/F-12 medium, made up to 500 mL. Wherein the fetal bovine serum is purchased from Gibco company, and the product number is 10099-141; the double antibody is purchased from Hyclone company, and the commodity number is SV 30010; advanced DMEM/F-12 medium was purchased from Gibco under the accession number 12634010.
And (3) after the preparation of the sample preservation solution is finished, subpackaging by using a 15mL centrifuge tube, wherein each tube contains 10mL, and the sample preservation solution can be stored for 1 month at 4 ℃ after subpackaging.
2. Sample cleaning solution
500mL of sample cleaning solution is prepared from the following raw materials: 100 × GlutaMAX, 5mL, final concentration 1 ×; 100 × double antibody of cyan chain, 5mL, and the final concentration is 1%; PBS, make up to 500 mL. Wherein 100 XGlutaMAX is purchased from Gibco, and the cargo number is 35050-; the green chain double antibody was purchased from Gibco under the cat number 15140122; PBS was purchased from Gibco under the cat # 20012027.
After the preparation of the sample cleaning solution, the sample cleaning solution is stored at 4 ℃ for 1 week.
3. Sample digestive juice
50mL of sample digestive juice is prepared from the following raw materials: collagenic II, final concentration 50 CDU; collagenic IV, the final concentration is 50 CDU; advanced DMEM/F-12 medium, made up to 50 mL. Wherein, the Collagenase II is purchased from Sigma company, and the product number is C6885; collagenase IV was purchased from Sigma under code number C5138; advanced DMEM/F-12 medium was purchased from Gibco, Inc. under the designation 12634010.
After the preparation of the sample digestive juice, sterilizing and filtering the sample digestive juice by using a 0.22 mu m filter, and subpackaging 0.5mL of frozen tube in 2mL of frozen tube, wherein the sample digestive juice can be stored for 6 months at the temperature of minus 80 ℃.
4. Sample stop solution
200mL of sample stop solution is prepared from the following raw materials: fetal bovine serum, 20mL, final concentration 10%; 100 × double antibody of cyan chain, 2mL, and 1% of final concentration; advanced DMEM/F-12 medium, make up to 200 mL. Wherein the fetal bovine serum is purchased from Gibco company, and the product number is 10099-141; the green chain double antibody is purchased from Gibco, with the product number of 15140122; advanced DMEM/F-12 medium was purchased from Gibco under the accession number 12634010.
After the preparation of the sample stop solution, the sample was sterilized and filtered through a 0.22 μm filter, and stored at 4 ℃ for 2 weeks.
Experimental example 2: obtaining colorectal cancer tissue sample
In cooperation with the hospital trimethyl, the cooperative development passed a formal medical ethical review. The attending physician selects patients to be grouped according to the TNM stage of the eighth edition of malignant tumor released by the International anti-cancer Union (UICC), and selects a proper sample for in vitro culture according to the clinical indication in the operation.
The attending physician provides basic clinical information about the patient's sex, age, medical history, family history, stage of pathology, clinical diagnosis, etc. The information related to the privacy of the patient, such as the name, the identification card number and the like of the patient, is hidden and replaced by a uniform experiment number, and the naming principle of the laboratory is region shorthand, sample receiving date and serial number. For example, the first sample received by Beijing at No. 4/5 of 2020, sample test number B20040501.
During the operation, a doctor collects a fresh sample in an operating room sterile environment, places the sample in a sample preservation solution prepared in advance according to the embodiment 1, and carries out cold storage transportation in 6 hours after the sample is separated to a laboratory for next operation.
Experimental example 3: pre-dissociation treatment of colorectal cancer tissue sample
The surgical instruments used in the following operations all need to be sterilized at high temperature and high pressure and can be used after being dried.
The experimental receiver performs sample information registration after receiving the information. The sample receiver hands over the sample information and the sample to the experimenter. The sample was washed 10 to 20 times with the sample washing solution of experimental example 1. And (4) removing excessive tissues such as connective tissues, fat tissues, necrotic tissues and the like in the sample by using a surgical instrument. The first sample is divided into three parts, the first part is fixed by formalin for staining, the second part is frozen at minus 80 ℃ for sequencing, the third part is used for organoid, and the samples are weighed.
Experimental example 4: digestion of colorectal cancer tissue samples
Surgical equipment used in the following operations needs high-temperature high-pressure sterilization and can be used after drying, and a centrifuge tube, a pipette and other consumable materials can be used after aseptic processing.
Adding 2mL of the digestive juice of the sample in Experimental example 1 per gram of the tissue sample, adding the digestive juice to the tissue sample, and shearing the tissue sample into 1mm by using an ophthalmic scissors3The left and right small blocks.
Digesting with shaking at 37 deg.C for 30min-2 h. Sample digestion was observed under the microscope every 30min until a large number of single cells were observed.
And (3) stopping digestion: the stop digest of Experimental example 1 was added in a volume 10 times the volume of the sample digest.
Sieving: the cell suspension was filtered through a sterile 40 μm filter to remove residual tissue.
Centrifuge at 500Xg for 5min at room temperature and discard the supernatant.
The cells were resuspended in 10mL sterile PBS, centrifuged at 500Xg for 5min at room temperature, and the supernatant discarded.
If erythrocytes are present, 2mL of erythrocyte lysate (from Solarbio, cat. No. R1010) can be added, lysis is carried out at room temperature for 3min, 10mL of sterile PBS buffer is added to stop lysis, centrifugation is carried out at 500Xg at room temperature for 5min, and the supernatant is discarded.
As shown in fig. 1, the dissociated single cell suspension contains a large amount of other types of cells, such as lymphocytes and fibroblasts, in addition to the primary tumor cells.
Experimental example 5: culture of colorectal cancer organoids
Resuspending the cells in the culture medium for colorectal organoid culture of example 2, observing the cell status under a microscope, counting the resuspended cells by a cytometer, inoculating the cells with the culture medium and matrigel at a volume ratio of 3:4, 40-60 μ L each gel drop, 25000 cells each drop, placing at 37 ℃ and 5% CO2Culturing under the condition.
The incubator is vertically placed for 5min and then reversely placed for 30min, and the culture solution for colorectal cancer organoid culture of example 2 is added for culture.
The cell status was observed every day, and the culture medium was changed every 3 days. If the culture medium turns yellow, the medium can be changed in advance until the cells form a cell mass larger than 80 μm.
The process of forming a cell pellet is followed.
As shown in FIG. 2, after 13d culture, cancer cells are amplified to form cell mass with diameter of 100 μm, and have intestinal structure, and total number of tumor cells can exceed 106Gel drop, the number of other types of cells gradually decreased or even disappeared. The culture solution of the invention is tested by a large number of samples, and the success rate of the in vitro culture of the primary tumor cells of the colorectal cancer can reach more than 90 percent.
As shown in fig. 3-7, a cell mass of the tracking swatter slowly grows up to form organoids with better intestinal structure, activity and form, which can be used for researching the occurrence and development mechanism of colorectal cancer, and organoids extracted and constructed from tumor tissues of patients with colorectal cancer are used for high-throughput drug screening, which is more beneficial to screening effective drugs.
Experimental example 6
Unlike Experimental example 5, Experimental example 6 uses the culture solution of comparative example 1 for the culture of colorectal cancer organoids, and the rest is the same.
As shown in fig. 9-13, following a bolus of cells in the beat, a bolus of cells was initially formed, but eventually no structural, multicellular organoids could be formed due to massive apoptosis, suggesting that icabet sodium is a factor required to maintain the highly active and intact state of colorectal cancer organoids.
Experimental example 7
Unlike Experimental example 5, Experimental example 7 uses the culture solution of comparative example 2 for the culture of colorectal cancer organoids, and the rest is the same.
As shown in FIGS. 14-17, following a bolus of cells, with the initial formation of the bolus and the eventual failure to form a structural, multicellular organoid, suggests that vitamin P4 plays an important role in maintaining and culturing colorectal organoids
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (3)

1. A culture solution for colorectal cancer organoid culture is characterized in that every 50mL of the culture solution is prepared from the following raw materials: incarbate sodium with a final concentration of 50 μ g/mL; vitamin P4 at a final concentration of 50 μ M; the final concentration of the human recombinant protein R-Spondin1 is 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 1 x final concentration; double resistance to the cyan chain, 1 x final concentration; n2 additive, 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- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole at a final concentration of 0.4 μ M; gastrin, final concentration 10 nM; a non-essential amino acid solution, final concentration 1 ×; nicotinamide, final concentration 5mM and Advanced DMEM/F-12 medium to 50 mL.
2. The culture solution for colorectal cancer organoid culture according to claim 1, wherein the double cyan-chain antibody contains 10000U/mL penicillin and 10mg/mL streptomycin.
3. A preparation method of a culture solution for colorectal cancer organoid culture is characterized by comprising the following steps:
the following raw materials are respectively weighed: icatsup sodium with a final concentration of 1-100 μ g/mL; vitamin P4 at a final concentration of 1-100. mu.M; the final concentration of the human recombinant protein R-Spondin1 is 30ng/mL-50 ng/mL; noggin, with final concentration of 80ng/mL-100 ng/mL; the final concentration of the human recombinant protein rhEGF is 8ng/mL-10 ng/mL; 4-hydroxyethyl piperazine ethanesulfonic acid at a final concentration of 1 mM; L-alanyl-L-glutamine at 1 x final concentration; double resistance to the cyan chain, 1 x final concentration; n2 additive, final concentration 1 ×; b27 additive, final concentration 1 ×; N-acetyl-L-cysteine at a final concentration of 0.09mM-0.125 mM; alk inhibitor A83-01 with final concentration of 0.3-0.5 μ M; 4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole at a final concentration of 0.3 μ M to 0.5 μ M; gastrin, final concentration 10 nM; a non-essential amino acid solution, final concentration 1 ×; nicotinamide with the final concentration of 5mM and the Advanced DMEM/F-12 culture medium being supplemented to 50mL, and the culture solution for culturing the colorectal cancer organoid is obtained after the raw materials are uniformly mixed.
CN202110109040.XA 2021-01-27 2021-01-27 Culture solution for colorectal cancer organoid culture and preparation method thereof Active CN112813028B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110109040.XA CN112813028B (en) 2021-01-27 2021-01-27 Culture solution for colorectal cancer organoid culture and preparation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110109040.XA CN112813028B (en) 2021-01-27 2021-01-27 Culture solution for colorectal cancer organoid culture and preparation method thereof

Publications (2)

Publication Number Publication Date
CN112813028A CN112813028A (en) 2021-05-18
CN112813028B true CN112813028B (en) 2022-07-15

Family

ID=75859639

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110109040.XA Active CN112813028B (en) 2021-01-27 2021-01-27 Culture solution for colorectal cancer organoid culture and preparation method thereof

Country Status (1)

Country Link
CN (1) CN112813028B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114181903A (en) * 2021-12-06 2022-03-15 浙江弘瑞医疗科技有限公司 Colorectal cancer organoid culture medium and stent-free 3D culture method
CN114591887B (en) * 2022-03-14 2022-10-14 杭州艾名医学科技有限公司 Breast cancer organoid culture solution, preparation method and culture method

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB201111244D0 (en) * 2011-06-30 2011-08-17 Konink Nl Akademie Van Wetenschappen Knaw Culture media for stem cells
CN110592020A (en) * 2018-06-13 2019-12-20 北京吉尚立德生物科技有限公司 Culture medium for culturing primary cells of colorectal cancer solid tumors

Also Published As

Publication number Publication date
CN112813028A (en) 2021-05-18

Similar Documents

Publication Publication Date Title
US20210338736A1 (en) Methods for generation of podocytes from pluripotent stem cells and cells produced by the same
CN112813028B (en) Culture solution for colorectal cancer organoid culture and preparation method thereof
US20180282691A1 (en) Hormone responsive tissue culture system and uses thereof
CN115279892A (en) Viable cell constructs for producing cultured dairy products and methods of use thereof
CN113736738B (en) Culture method of gastric cancer micro-tumor cell model
CN108504625B (en) Mouse fibroblast and application thereof
EP1104993B1 (en) C3a serum-free clonal cell line and methods of use
CN116024159A (en) Method for constructing murine skin organoids
Zhang et al. A pre‑clinical model combining cryopreservation technique with precision‑cut slice culture method to assess the in vitro drug response of hepatocellular carcinoma
CN113755441A (en) Culture method of lung cancer micro-tumor cell model
US20220213433A1 (en) Three-Dimensional Cross-Linked Scaffolds of Cord Blood Plasma and Their Use
CN112481193A (en) Standardized culture medium for three-dimensional culture of intestinal and intestinal cancer tissue organoid and culture method
CN115261326B (en) Culture medium and culture method for establishing breast cancer and paracancestral organ model
CN116590232A (en) Thyroid cancer organoid, culture medium and culture method
CN110607278A (en) Tumor cell culture and application thereof
CN115851577A (en) Method for constructing osteosarcoma organoid model based on single cell
CN116536264A (en) Special serum-free culture medium for colon cancer organoids
CN115369087A (en) Culture medium and culture method of primary liver cancer cells
CN115058394B (en) Adenoid cystic carcinoma organoid model, construction method and application thereof
WO2000075287A2 (en) Long term cell culture of human carcinoma
US20210062157A1 (en) Methods and compositions for culturing alveolar cells
CN118272310A (en) Isolation culture method of primary cells of human colorectal cancer and detection method of drug sensitivity
Wu Growth of human lung tumor cells in culture
CN117467616B (en) Special culture medium for preparing oral cancer micro-tumor model, related method product and application
CN117757721A (en) Culture medium for lung cancer organoids and culture method for lung cancer organoids

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant