CN116769714A - Preparation method of organoid model - Google Patents

Preparation method of organoid model Download PDF

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Publication number
CN116769714A
CN116769714A CN202210241427.5A CN202210241427A CN116769714A CN 116769714 A CN116769714 A CN 116769714A CN 202210241427 A CN202210241427 A CN 202210241427A CN 116769714 A CN116769714 A CN 116769714A
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organoid
culture
cancer cells
tissue
concentration
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王珂
于敏
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Tianjin Medical University Cancer Institute and Hospital
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Tianjin Medical University Cancer Institute and Hospital
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    • 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
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    • 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
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
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Abstract

The invention discloses a preparation method of an organoid model. The method can promote the rapid growth of the tumor organoids, shorten the culture time, solve the problem of uneven organoids, and realize multi-layer organoid culture.

Description

Preparation method of organoid model
Technical Field
The invention relates to the technical field of cell biology, in particular to a preparation method of an organoid model.
Background
Disease models or materials are more reflective of physiological tumor studies that have long been looking for features of real patient tumors. To better generalize the cancer of patients, various disease models have been developed. The in vitro culture of the primary cells growing in 2D loses the form in the tumor body, the three-dimensional (3D) culture of the model cell line lacks the characteristic of the heterogeneity of the parent body, the tumor organoid invented by the primary tumor cell culture technology depending on the origin of the patient maintains the molecular, genetic and pathological characteristics of the primary tumor, has the advantages of in vivo tumor drug sensitivity and in vitro traditional drug sensitivity test, and provides a better model for the individuation treatment of the tumor.
However, in the prior art, there is also a problem of low success rate of culture of tumor organoids, so that a tumor organoid model with high success rate of cell culture needs to be found.
Disclosure of Invention
The invention provides a preparation method of a organoid model, which aims at the problem of low success rate of tumor organoid culture in the prior art.
The technical scheme provided by the invention is as follows:
a method for preparing an organoid model comprising the steps of:
step 1) obtaining a cancer cell sample;
step 2) collecting cancer cells from the sample obtained in step 1);
step 3) re-suspending the cancer cells collected in the step 2) by using 3 times concentration organoid culture medium, uniformly mixing the cancer cells with 2 times of the volume of the culture medium, spreading the formed dropping liquid in a culture container, and standing at 37 ℃ for 15-20 minutes;
step 4) adding the organoid culture solution with the concentration of 1 time as much as the volume of the hydrogel drop solution obtained in the step 3), and placing the organoid culture solution into 5% CO at 37 DEG C 2 Culturing in incubator, and changing organoid culture solution with concentration of 1 time every 3 days until cancer cells grow to proper size;
wherein the 1-fold concentration organoid culture solution comprises one or more of 1-15 ng/mL of epidermal growth factor, 2-30 ng/mL of fibroblast growth factor, 2-20 ug/mL of insulin, 0.1-0.5 mM of vitamin E, 0.1-2 uM of estradiol or 2-25 ng/mL of hydrocortisone.
Preferably, the cancer cell sample in step 1) is tumor tissue or ascites.
Preferably, the cancer cell sample in step 1) is a tumor tissue, and the collecting method in step 2) is to digest the tumor tissue, and the tissue digestive juice used in the digestion contains collagenase I, collagenase IV, dispase or pancreatin.
Preferably, the tissue digestion solution comprises collagenase I200-500U/mL, deoxyribonuclease 75-200U/mL and CaCl 2 1-3mM, formulated with HBSS.
Preferably, the density of the cancer cells in step 3) is 1×10 4 ～6×10 4 Individual cells/mL.
Preferably, the organoid culture solution of step 3) further comprises D-glucose, sodium bicarbonate, sodium pyruvate, HEPES, L-glutamine, a 1% mixture of green streptomycin and RPMI1640 medium containing 5-10% fetal bovine serum.
Preferably, the organoid culture solution in step 3) further comprises sodium bicarbonate, sodium pyruvate, L-glutamine, a 1% mixture of green streptomycin and a high sugar DMEM medium containing 5-10% fetal bovine serum.
Preferably, the cancer cell is an ovarian cancer cell.
In another aspect, the present invention provides an organoid prepared by the above preparation method.
In another aspect, the invention provides the use of the organoids described above in the culture of cancer cells.
The beneficial effects of the invention are as follows:
the method can promote the rapid growth of the tumor organoids, shorten the culture time, solve the problem of uneven organoids, and realize multi-layer organoid culture.
Drawings
FIG. 1 is a graph showing the results of hydrogel culturing ovarian cancer tissue organoids for 4 days, wherein A is the hydrogel culture of key ingredient 1; b, hydrogel culture of key component 2; c, hydrogel culture of a key component 3; d, hydrogel culture of key component 4; e, hydrogel culture of key component 5; f, hydrogel culture of a key component 6; hydrogel culture of organoid medium at G1-fold concentration; h hydrogel culture without any critical components; i organoid medium culture at 1-fold concentration in anhydrous gel.
Detailed Description
The invention discloses a preparation method of an organoid model, and a person skilled in the art can refer to the content of the description and properly improve the implementation of technological parameters. It is to be particularly pointed out that all similar substitutes and modifications apparent to those skilled in the art are deemed to be included in the invention and that the relevant person can make modifications and appropriate alterations and combinations of what is described herein to make and use the technology without departing from the spirit and scope of the invention.
In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or components. The terms "such as," "for example," and the like are intended to refer to exemplary embodiments and are not intended to limit the scope of the present disclosure.
In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail with reference to specific embodiments.
Example 1: construction of organoids derived from ovarian cancer tissue
1. Collection of ovarian tissue samples
The information table of sample collection is filled in advance by the operating doctor before operation, and the content of the information table comprises the name, age, clinical diagnosis, whether the patient is subjected to chemotherapy before operation, operation time, tumor type, whether the tumor sample is agreed to be cultured in vitro, tested in vitro and the like. After the rapid frozen section in the operation is diagnosed as the epithelial ovarian cancer, under the condition of ensuring that the pathological diagnosis after the operation is not influenced, a surgeon cuts fresh tumor tissue with better quality under the aseptic condition, and the minimum size of 0.5cm x 0.5cm x 0.5cm is about 0.5cm x 0.5cm x 0.5cm in each case, and the tumor tissue is aseptically transported to a laboratory within 6 hours.
2. Tissue treatment before tissue organoid culture of ovarian cancer
Asepsis operation is carried out in an ultra-clean workbench, ovarian cancer tissues are cut into 1-3mm sizes by using an aseptic ophthalmic scissors or a blade in a 6cm cell culture dish, the ovarian cancer tissues are transferred to a 15mL centrifuge tube, the tissue is centrifugally cleaned by using HBSS containing double antibodies, the cleaning liquid is discarded, tissue digestion liquid is added into a tissue block, and 1mL of tissue digestion liquid is added into every 100mg of tissues, wherein the components are as follows: 200-500U/mL collagenase I, 75-200U/mL deoxyribonuclease and CaCl 2 1-3mM, formulated with HBSS, incubated on a shaker at 37℃and digested for 0.5-3h depending on the amount of tissue mass.
The primary ovarian cancer cell yield obtained by treating the same amount of ovarian cancer tissue with different digestive enzymes under the same conditions is compared with collagenase I > collagenase IV > dispase > pancreatin.
3. Construction and culture of ovarian cancer tissue organoid model
Visual observation of the absence of hard lump-like tissue in 15mL centrifuge tube, adding equal amount of HBSS in digestive tissue liquid to stop digestion, centrifuging at 1000rpm for 5min at 4deg.C, collecting separated cancer cells, re-suspending with ovarian cancer organoid culture medium, centrifuging, and precipitating with 1×10 4 -6×10 4 Inoculating the culture medium into a proper pore plate, culturing with non-animal-derived hydrogel, re-suspending primary cells with 3 times concentration organoid culture medium (1 time concentration organoid culture medium is shown in Table 1), mixing with 2 times volume of hydrogel to form dripping solution, spreading in pore plate or culture dish, standing at 37deg.C for 15min, adding equal volume of 1 time concentration organoid culture medium, and placing into 37deg.C 5% CO 2 Culturing in incubator.
The liquid is changed once every 3 days, and organoids with proper sizes can be grown for 3-10 days for other experiments. The results are shown in FIG. 1. The success rate of tissue organoid culture was 83.3%. The calculation formula is as follows: tissue organoid culture success rate = number of tissue organoid culture success rate ≡total number of ovarian cancer specimens (including surgical specimens, endoscopic specimens and puncture specimens) ×100%
Table 11 times concentration organoid medium main ingredient formulation
Key components English name Chinese name Working concentration
1 EGF Epidermal growth factor 1-15ng/mL
2 FGF Fibroblast growth factor 2-30ng/mL
3 Insulin Insulin 2-20ug/mL
4 VE Vitamin E 0.1-0.5mM
5 Estradiol Estradiol as a pharmaceutical 0.1-2uM
6 Hydrocortisone Hydrocortisone 2-25ng/mL
Is prepared with RPMI1640 medium containing D-glucose, sodium bicarbonate, sodium pyruvate, HEPES, L-glutamine, 1% green streptomycin mixed solution and 5-10% fetal bovine serum. Or high sugar DMEM medium containing sodium bicarbonate, sodium pyruvate, L-glutamine, 1% green streptomycin mixed solution and 5-10% fetal calf serum.
Example 2: construction of organoids derived from ascites due to ovarian cancer
1. Collection of ovarian ascites samples
The medical practitioner fills the sample collection information table in advance before collection, and the content of the sample collection information table comprises the name, age and clinical diagnosis of a patient, whether the patient receives radiotherapy and chemotherapy, the operation time, the tumor type, whether the tumor sample is agreed to be cultured in vitro, the in vitro test agent and the like. The operation doctor samples the ward and nurses samples the ward, 50-100mL of ascites is extracted under the condition of ensuring that pathological diagnosis is not affected, and the ascites is aseptically transported to a laboratory within 6 hours.
2. Ovarian cancer ascites organoid culture
And (3) performing aseptic operation in a laboratory, centrifuging at 1000rpm for 5min to collect cancer cells of ascites, centrifuging and cleaning once by using HBSS or PBS containing double antibodies, discarding the cleaning solution, adding the split red solution into cell sediment, adding 1mL of the split red solution into every 50mL of ascites, uniformly mixing and standing for 5min, centrifuging and collecting the cell sediment, centrifuging and cleaning once by using HBSS or PBS, and inoculating and culturing the same way as ovarian cancer tissue organoids. The success rate of the culture of the ovarian cancer ascites organoid is 100 percent. The calculation formula is as follows: success rate of ascites organoid culture = number of successful cases of cultured ascites organoid ≡total number of cases of cultured ovarian cancer × 100%
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. A method for preparing an organoid model, comprising the steps of:
step 1) obtaining a cancer cell sample;
step 2) collecting cancer cells from the sample obtained in step 1);
step 3) re-suspending the cancer cells collected in the step 2) by using 3 times concentration organoid culture medium, uniformly mixing the cancer cells with 2 times of the volume of the culture medium, spreading the formed dropping liquid in a culture container, and standing at 37 ℃ for 15-20 minutes;
step 4) adding water obtained in step 3)Gel drop solution of organoid culture solution with concentration of 1 times of the same volume, and adding 5% CO at 37deg.C 2 Culturing in incubator, and changing organoid culture solution with concentration of 1 time every 3 days until cancer cells grow to proper size;
wherein the 1-fold concentration organoid culture solution comprises one or more of 1-15 ng/mL of epidermal growth factor, 2-30 ng/mL of fibroblast growth factor, 2-20 ug/mL of insulin, 0.1-0.5 mM of vitamin E, 0.1-2 uM of estradiol or 2-25 ng/mL of hydrocortisone.
2. The method of claim 1, wherein the cancer cell sample of step 1) is a tumor tissue or ascites.
3. The method of claim 2, wherein the cancer cell sample in step 1) is a tumor tissue, and the collecting in step 2) is a digestion of the tumor tissue, and the digestion uses a tissue digestion solution comprising collagenase I, collagenase IV, dispase or pancreatin.
4. The method according to claim 3, wherein the tissue digestion solution comprises collagenase I200-500U/mL, deoxyribonuclease 75-200U/mL and CaCl 2 1-3mM, formulated with HBSS.
5. The method of claim 1, wherein the density of the cancer cells in step 3) is 1X 10 4 ～6×10 4 Individual cells/mL.
6. The method of claim 1, wherein the organoid culture broth of step 3) further comprises D-glucose, sodium bicarbonate, sodium pyruvate, HEPES, L-glutamine, a 1% mixture of green streptomycin, and RPMI1640 medium containing 5-10% fetal bovine serum.
7. The method according to claim 1, wherein the organoid culture solution of step 3) further comprises sodium bicarbonate, sodium pyruvate, L-glutamine, a 1% mixture of green streptomycin and a high sugar DMEM medium containing 5 to 10% fetal calf serum.
8. The method according to any one of claims 1 to 7, wherein the cancer cell is an ovarian cancer cell.
9. A organoid prepared by the preparation method as claimed in any one of claims 1 to 7.
10. Use of the organoid of claim 9 in culturing cancer cells.
CN202210241427.5A 2022-03-11 2022-03-11 Preparation method of organoid model Pending CN116769714A (en)

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