CN110396501B - Three-dimensional spheroid culture method for maintaining dryness of breast cancer stem cells in vitro - Google Patents
Three-dimensional spheroid culture method for maintaining dryness of breast cancer stem cells in vitro Download PDFInfo
- Publication number
- CN110396501B CN110396501B CN201910707496.9A CN201910707496A CN110396501B CN 110396501 B CN110396501 B CN 110396501B CN 201910707496 A CN201910707496 A CN 201910707496A CN 110396501 B CN110396501 B CN 110396501B
- Authority
- CN
- China
- Prior art keywords
- breast cancer
- cancer stem
- cell
- stem cells
- culture
- 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
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
- C12N5/0695—Stem cells; Progenitor cells; Precursor 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
- 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
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- 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
- C12N2501/30—Hormones
- C12N2501/33—Insulin
-
- 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
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
-
- 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
- C12N2513/00—3D culture
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Oncology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a three-dimensional spheroid culture method for maintaining the dryness of breast cancer stem cells in vitro. Adopting a rotary bioreactor to carry out three-dimensional spheroid culture on the human breast cancer stem cell to directly obtain a breast cancer stem cell spheroid, wherein the obtained cell is ALDH1+/CD44+/CD24‑The phenotype is a recognized breast cancer stem cell phenotype, and the method is a reliable breast cancer stem cell in-vitro culture method. The method is simple to operate, and compared with the prior art such as a low-adhesion material method, a pendant drop method, a gel method and the like, the method greatly improves the balling rate of the stem cells, prolongs the dry maintenance time to at least 21 days, can perform large-scale amplification culture, and greatly reduces the cost in the aspects of reagent consumables, human resources and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a three-dimensional spheroid culture method for maintaining the dryness of breast cancer stem cells in vitro.
Background
Early breast cancer (grade I, II, IIIA and operable IIIC) is treated primarily with surgical intervention and radiotherapy. Adjuvant chemotherapy regimens, represented by hormone preparations, trastuzumab and cytotoxic chemotherapeutic agents, are used to prevent the formation of micrometastases after surgery. Grade IIIB and non-surgical grade IIIC generally use systemic chemotherapy or hormone therapy to degrade control of the primary focus prior to surgical or radiotherapeutic treatment. Although the above method is effective in reducing the volume of tumor mass in a short period of time, if cancer stem cells cannot be completely eliminated, the drug effect must not be maintained for a long period of time, and there is a high possibility that the tumor will be resistant to drugs and recur. Therefore, breast cancer stem cells are crucial for in vitro drug screening.
Cancer Stem Cells (CSCs) are closely associated with tumor growth, recurrence, and metastasis, and are resistant to most of today's chemoradiotherapy approaches. Quantitatively, it accounts for about 5.0% of the total number of cancer cells; qualitatively, like normal adult stem cells, have the ability to self-renew and differentiate in multiple cells, specifically expressing acetaldehyde dehydrogenase 1(ALDH 1).
CSCs achieve tumor heterogeneity by virtue of self-replicating processes that asymmetrically produce clonal and non-clonal differentiated progeny. Since 2003 Al-Hajj reported to possess CD44 for the first time+/CD24-/lowAfter the phenotypic breast cancer cells were CSCs, the scholars agreed that only CD44 was present+/CD24-/lowCan form secondary (recurrent or metastatic) tumors. It has also been reported that only 20 CDs 44 are provided+/CD24-/ALDH1+The breast cancer CSC can form tumors in mice, and the three marked cells in the group have the strongest tumorigenicity.
Mammary CSCs differ from the differentiated terminal cells that undergo anoikis, which spontaneously aggregate to form mammary spheroids (mammospheres) and continue to proliferate in an in vitro suspension state. The transforming growth factor-beta 1 as one of the important signal transduction pathway members related to the CSC self-replication can up-regulate the mammosphere forming ability of mammary epithelial cells by 40 times, and finally realize the EMT. Another study showed that EMT-completed cancer cells were metastasized by "CTC clusterics" rather than single cells as a mobile unit, leaving the primary foci through the systemic circulation to the distant site. A cancer focus is a tissue with a three-dimensional spatial structure. Thus, the spontaneous formation of mammary spheroids by CSCs is one of its important features, and is also a critical biological stage that they must undergo in order to achieve survival, proliferation and micrometastases.
Simulating the in vivo cancer stem cell microenvironment is the research direction for the stem dryness maintenance of cancer stem cells cultured in vitro. The three-dimensional spheroid culture is a natural interaction between cells or matrixes to form natural micro tissues which are closer to the in-vivo three-dimensional microenvironment of cancer stem cells. In recent years, researches show that the in-vivo microenvironment of the cancer stem cells can be better simulated by adopting a three-dimensional spheroid culture method such as a low-adhesion material method, a pendant drop method, a gel method and the like than two-dimensional culture, and the dryness characteristics of the cancer stem cells are more effectively maintained. However, these three methods have their limitations: firstly, at least 10 days are needed for forming usable mammary glands; secondly, the phenotype and the proportion of the cancer stem cells are difficult to maintain for a long time, the proportion of the stem cells cultured in vitro is gradually reduced (cell differentiation) along with the number of passages, and the stem cells cannot be used after about 14 days; and thirdly, large-scale amplification culture cannot be realized, and the difficulty of subsequent research is high. The galactophore suspension culture method disclosed in the article "culture and identification of breast cancer stem cell" published by Zhengwenbo et al belongs to the specific application of the suspension drop method. As described herein: maintaining an undifferentiated state of cancer stem cells therein using DMEM/F12 to which growth factors are added, but no name of the growth factors is specifically given; the DMEM/F12 culture medium is a general culture medium for in vitro cell culture, and the special culture medium for serum-free mammary epithelial cells is used, so that the special culture medium is more suitable for breast cancer stem cells; ③ using Western blotting to detect the obvious increase of the protein concentration of the mammosphere ALDH1 after culturing for 10-14 days in vitro, using CD44 and CD24 antibodies to carry out flow cytometry detection, and obtaining CD44 by the suspension culture method given by the authors+/CD24-/lowThe proportion of cancer stem cells is 62.36%. According to published authoritative literature (Ginester, et al. ALDH1 is amarker of normal and malignant human breast cells and a predictor of clinical out-of-Cell. Cell Stem Cell,2007,1(5): 555-. The authors of the present invention digested tumor tissue of breast cancer patients into single cells at 1X 104Suspension culture in ml to obtain mixed spheroids of cancer stem cells and differentiated cancer cellsThe initial proportion of stem cells should not exceed 10%, and the proportion of cancer stem cells cultured in vitro is only reduced, so the literature data is to be studied.
In addition, the breast cancer stem cells are easy to differentiate in vitro culture, the dryness maintenance of the breast cancer stem cells is always a problem to be solved by in vitro culture of the breast cancer stem cells, the dryness maintenance is not involved in the culture of the non-stem cells, and if the culture method of the non-stem cells is directly applied to the culture of the breast cancer stem cells, the dryness of the breast cancer stem cells cannot be maintained. Therefore, the experience and system of non-stem cell culture cannot be used as reference for breast cancer stem cell culture.
Disclosure of Invention
The invention aims to overcome the defect that the dryness of breast cancer stem cells cannot be maintained for a long time in the prior art, and provides a three-dimensional spheroid culture method for maintaining the dryness of breast cancer stem cells in vitro based on a rotary bioreactor.
The technical scheme adopted by the invention is as follows:
a three-dimensional spheroid culture method for maintaining the dryness of breast cancer stem cells in vitro comprises the following steps:
1) adding human breast cancer stem cells in a single-cell state into a stem cell culture medium to prepare a cell suspension;
2) adding cell suspension into a rotary bioreactor, and setting the rotating speed of the cell suspension;
3) placing the rotary bioreactor into a cell culture box for culture, and replacing a stem cell culture medium once every 2-4 days;
4) culturing for 2-7 days to obtain the breast cancer stem cell spheroids with proper sizes.
Preferably, in step 1), the human breast cancer stem cells used are cancer stem cells obtained by digesting and sorting fresh breast cancer tissues in an operation.
Preferably, in step 1), the cell density of the cell suspension is 1 × 104～1×105one/mL.
Preferably, the stem cell culture medium is added with 10-20 ng/mL epidermal growth factor EGF, 10-20 ng/mL basic fibroblast growth factor bFGF, 5-10 mug/mL Insulin Insulin and 10-6M hydrocortisone, 80-150U/mL penicillin, 80-150 mg/mL streptomycin serum-free special culture medium for mammary epithelial cells (
MammoCultTMMedium)。
Preferably, in step 2), the rotary bioreactor is manufactured by SYNTHECON.
Preferably, in the step 2), the rotating speed of the rotary bioreactor is 15-25 rpm.
Preferably, in step 3), the culture conditions are: 4.9-5.1% CO at 37.0-37.5 deg.C2And saturation humidity.
The invention has the beneficial effects that:
the invention adopts a rotary bioreactor to carry out three-dimensional spheroid culture on human breast cancer stem cells, and directly obtains breast cancer stem cell spheroids, wherein the obtained cells are ALDH1+/CD44+/CD24-The phenotype is a recognized breast cancer stem cell phenotype, and the method is a reliable breast cancer stem cell in-vitro culture method. The method is simple to operate, and compared with the prior art such as a low-adhesion material method, a pendant drop method, a gel method and the like, the method greatly improves the balling rate of the stem cells, prolongs the dry maintenance time to at least 21 days, can perform large-scale amplification culture, and greatly reduces the cost in the aspects of reagent consumables, human resources and the like.
Drawings
FIG. 1 shows spheroids of breast cancer stem cells formed by culturing in example 1.
A is a human breast cancer stem cell sphere which is cultured and formed by a rotary reactor and observed under a mirror for 2 days; and B, observing a human breast cancer stem cell sphere formed by culturing in a rotary reactor under a mirror for 7 days.
Fig. 2 shows the results of flow cytometry for ALDH1, CD44, and CD 24.
A is the detection result of flow cytometry by carrying out three-dimensional spheroid culture on human breast cancer stem cells for 14 days; and B is the detection result of flow cytometry by carrying out three-dimensional spheroid culture on the human breast cancer stem cells for 21 days.
As shown in the figure, adoptThree-dimensional spheroid culture of human breast cancer stem cells in a rotating bioreactor for 14 days and 21 days, and then culturing the stem cells (ALDH1)+/CD44+/CD24-Phenotype) ratio was 86% and 45%, respectively.
FIG. 3 shows the subcutaneous tumorigenesis of nude mice by breast cancer stem cell spheroids obtained by 21 days of in vitro culture in a rotating bioreactor.
Detailed Description
The breast cancer stem cells used in the examples are cancer stem cells obtained by digesting and sorting fresh breast cancer tissues in the operation.
Example 1
1. Separating mammary gland tissues:
1) placing fresh mammary tissue into a precooled culture medium containing 5% fetal bovine serum EpiCult-B;
2) transferring the tissue into a sterile glass culture dish, crushing by a scraper, and transferring into a tissue digestion bottle;
3) preparing EpiCult-B Medium digestive juice containing collagenase/hyaluronidase, adding into a digestive flask to ensure that the tissue block is completely immersed and suspended, and sealing by sterile tinfoil paper;
4) horizontally oscillating at 37 ℃ for digestion until large tissue blocks disappear;
5) transferring the digestive juice to a sterile 50mL centrifuge tube, centrifuging at 80g for 30 seconds;
6) discarding the uppermost fat layer;
7) and centrifuging the mixture for 3 minutes at 200g in another sterile 50mL centrifuge tube, and precipitating the mixture into mammary epithelial cells.
2. Isolation of single cell suspension:
1) adding 3mL of preheated pancreatin containing EDTA into the precipitate, and blowing and beating the gun head for 1 minute;
2) 10mL of cold Hanks solution containing 2% fetal bovine serum was added, 350g was added, and centrifugation was performed for 5 minutes;
3) the supernatant was discarded as much as possible;
4) 2mL of pre-warmed neutral protease (5mg/mL) and 200. mu.L of DNase I (1mg/mL) were added, and the pipette tip was blown for 1 minute;
5) the cell suspension was diluted by adding 10mL of cold Hanks solution containing 2% fetal bovine serum and filtered through a 40 μm filter to a new sterile medium50mL centrifuge tube, 350g, 5 minutes centrifugation, and abandoning the supernatant, adding PBS to resuspend the pellet, adjusting the cell concentration to 1X 106one/mL.
3. Preparing an ALDH1 detection sample:
1) 2 EP tubes were removed, labeled "test" and "control", respectively;
2) taking 1mL of cell suspension to a test tube;
3) add 5. mu.L DEAB reagent to the "control" tube;
4) adding 5 μ L of AlDEFUOR reagent into the "test" tube, rapidly mixing, sucking 500 μ L into the "control" tube, and immediately mixing;
5) placing the "test" and "control" into a cell incubator and incubating for 60 minutes;
6) centrifugation at 250g for 5 min;
7) each of the "test" and "control" was resuspended in 200. mu.L of PBS.
Markers CD44 and CD24 and flow cytometry sorting:
1) adding 5 mu L of each of CD44-APC and CD24-PE antibodies to a test tube, and incubating for 15 minutes at room temperature in a dark place;
2) adding 1mL of PBS respectively, centrifuging at 1000rpm for 5 minutes, and removing supernatant;
3) 1mL of PBS was added for each resuspension, sorting was performed using a flow cytometer (BD Aria), and the sorted cells were collected in stem cell culture medium.
4. Culturing spheroids:
1) adding stem cell culture medium to resuspend cells, adjusting cell density to 1 × 104Per mL; the stem cell culture medium is added with 20ng/mL epidermal growth factor EGF, 20ng/mL basic fibroblast growth factor bFGF, 10 mug/mL Insulin Insulin and 10-6Serum-free special culture medium for mammary epithelial cells containing M hydrocortisone, 150U/mL penicillin and 150mg/mL streptomycin (
MammoCultTMMedium)。
2) Adding the cell suspension into a rotary bioreactor; loading a rotary bioreactor, and setting the rotating speed of the rotary bioreactor to be 20 revolutions per minute;
3) placing the rotary bioreactor into a cell culture box for culture, and replacing a stem cell culture medium once every 3 days;
4) the next day of culture, human breast cancer stem cell spheroids were observed under the mirror as shown in fig. 1A. The visible small cell mass is circular/elliptical, the boundary is clear, the quality is solid, the cells in the sphere are uniformly distributed, and the sphere are independent. Along with cell division, the sphere gradually becomes bigger, is smooth and round, and has a more compact internal structure and strong refractivity. Cultured for 7 days, and the human breast cancer stem cell spheres were observed under a mirror, as shown in fig. 1B.
Example 2
1. Mammary gland spheroid digestion in vitro culture for 14 days:
1) collecting the breast spheroids which are cultured for 14 days to a 50mL centrifuge tube, centrifuging for 5 minutes at 350g, and discarding the supernatant as much as possible;
2) 1mL of pre-heated EDTA-containing pancreatin is added, the range of a sample adding gun is slightly adjusted to be small, the gun head is wetted, and foam is avoided;
3) the gun head props against the pipe wall, blows the mammary gland spheroid up and down gently, and washes the pipe wall;
4) 5mL of precooled Hanks solution containing 2% fetal calf serum is added, 350g is added, and centrifugation is carried out for 5 minutes;
5) the supernatant was discarded, 1mL of PBS was added to resuspend the pellet, and the cell concentration was adjusted to 1X 105One part was subjected to subsequent experiments and the other part was subcultured.
2. Preparing an ALDH1 detection sample:
1) 2 EP tubes were removed, labeled "test" and "control", respectively;
2) taking 1mL of cell suspension to a test tube;
3) add 5. mu.L DEAB reagent to the "control" tube;
4) adding 5 μ L of AlDEFUOR reagent into the "test" tube, rapidly mixing, sucking 500 μ L into the "control" tube, and immediately mixing;
5) placing the "test" and "control" into a cell incubator and incubating for 60 minutes;
6) centrifugation at 250g for 5 min;
7) each of the "test" and "control" was resuspended in 200. mu.L of PBS.
3. Markers CD44 and CD24 and flow cytometry detection:
1) adding 5 mu L of each of CD44-APC and CD24-PE antibodies to a test tube, and incubating for 15 minutes at room temperature in a dark place;
2) adding 1mL of PBS respectively, centrifuging at 1000rpm for 5 minutes, and removing supernatant;
3) each 200. mu.L of PBS was added for resuspension and detection was performed using a flow cytometer (BD Calibur).
As shown in FIG. 2A, the stem cell of breast cancer cultured in vitro for 14 days was ALDH1+/CD44+/CD24-Phenotype, accounting for still more than 80%. Another portion of the untreated cells were passaged:
1) adding stem cell culture medium to resuspend cells, adjusting cell density to 1 × 104Per mL;
2) adding the cell suspension into a rotary bioreactor; loading a rotary bioreactor, and setting the rotating speed of the rotary bioreactor to be 15 r/min;
3) placing the rotary bioreactor into a cell culture box for culture, and replacing a stem cell culture medium once every 3 days;
4) the culture was continued for 7 days.
Digesting the human breast cancer stem cell spheres cultured in vitro for 21 days, preparing an ALDH1 detection sample, marking CD44 and CD24 and performing flow cytometry detection. As a result, as shown in FIG. 2B, the stem cell of breast cancer cultured in vitro for 21 days was ALDH1+/CD44+/CD24-Phenotype, the proportion is still around 45%.
Example 3
Subcutaneous open cell tumor:
1) dividing the same batch of 4-week-old NOD/SCID mice into three groups, 2 mice/group;
2) pouring the breast cancer stem cell spheroids obtained by culturing 21 days in vitro in the rotary bioreactor into a 10cm cell culture dish from the rotary bioreactor, respectively mixing three small, medium and large particles with matrigel, and inoculating the mixture to the middle and rear parts of right axilla;
3) three weeks later, the mice were sacrificed and the tumor bodies were removed for sizing.
The results are shown in fig. 3, and the spheroids of breast cancer stem cells expanded in vitro can form tumors subcutaneously in nude mice, indicating that the sternness is maintained.
Claims (2)
1. A three-dimensional spheroid culture method for maintaining the dryness of breast cancer stem cells in vitro is disclosed, wherein the breast cancer stem cells are obtained by digesting and sorting fresh breast cancer tissues in an operation and have the phenotype of ALDH1+/CD44+/CD24-The method is characterized by comprising the following steps:
1) adding human breast cancer stem cells in a single-cell state into a stem cell culture medium to prepare a cell suspension; the human breast cancer stem cell is obtained by digesting and sorting fresh breast cancer tissues in an operation, and the cell density in the cell suspension is 1 multiplied by 104～1×105The stem cell culture medium is added with 10-20 ng/mL epidermal growth factor EGF, 10-20 ng/mL basic fibroblast growth factor bFGF, 5-10 mug/mL Insulin and 10-6Serum-free special culture medium STEMCELL for mammary epithelial cells, wherein the serum-free special culture medium comprises M hydrocortisone, 80-150U/mL penicillin and 80-150 mg/mL streptomycin®MammoCultTMMedium；
2) Adding a cell suspension into a rotary bioreactor, and setting the rotating speed of the cell suspension to be 15-25 revolutions per minute;
3) placing the rotary bioreactor into a cell culture box for culture under the following culture conditions: 4.9-5.1% CO at 37.0-37.5 deg.C2Changing the stem cell culture medium once every 2-4 days at saturated humidity;
4) culturing for 2-7 days to obtain the breast cancer stem cell spheroids with proper sizes.
2. The method of claim 1, further comprising: in step 2), the rotary bioreactor is manufactured by SYNTHECON.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910707496.9A CN110396501B (en) | 2019-08-01 | 2019-08-01 | Three-dimensional spheroid culture method for maintaining dryness of breast cancer stem cells in vitro |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910707496.9A CN110396501B (en) | 2019-08-01 | 2019-08-01 | Three-dimensional spheroid culture method for maintaining dryness of breast cancer stem cells in vitro |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110396501A CN110396501A (en) | 2019-11-01 |
| CN110396501B true CN110396501B (en) | 2020-10-30 |
Family
ID=68327013
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910707496.9A Active CN110396501B (en) | 2019-08-01 | 2019-08-01 | Three-dimensional spheroid culture method for maintaining dryness of breast cancer stem cells in vitro |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110396501B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116496974A (en) * | 2022-01-19 | 2023-07-28 | 合肥中科普瑞昇生物医药科技有限公司 | Culture medium, culture method and application of mammary epithelial cells |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101050452A (en) * | 2007-03-27 | 2007-10-10 | 江苏省人民医院 | Method for separating stem cell of breast cancer |
| CN105274059A (en) * | 2015-06-15 | 2016-01-27 | 中山大学肿瘤防治中心 | Two breast cancer stem cell lines capable of being stably cultured and keeping original characteristics, separation method and uses thereof |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103266083A (en) * | 2013-05-23 | 2013-08-28 | 成都美进生物科技有限公司 | Culture method of breast cancer stem cell |
| CN103396995B (en) * | 2013-07-16 | 2015-09-23 | 广州赛哲生物科技有限公司 | A kind of three-dimensional culture method screening breast carcinoma stem cell |
| CN105087494A (en) * | 2015-09-11 | 2015-11-25 | 中国人民解放军第四军医大学 | Culture method of breast cancer stem cells |
| CN106635997A (en) * | 2016-12-19 | 2017-05-10 | 南京千年健干细胞基因工程有限公司 | Reagent and kit for enriching breast cancer stem cells from breast cancer cell strains |
| CN106386787A (en) * | 2016-12-19 | 2017-02-15 | 南京千年健干细胞基因工程有限公司 | Breast cancer stem cell cryopreservation protection agent and prepared cryopreservation protection kit |
| US10100275B2 (en) * | 2017-03-16 | 2018-10-16 | King Faisal Specialist Hospital & Research Centre | Method for generating human multipotent mammary stem cells from normal primary breast luminal cells |
| CN109652374A (en) * | 2019-02-27 | 2019-04-19 | 赛璟生物医药科技(上海)有限公司 | It is a kind of for maintaining the serum free medium of neural stem cell in vitro culture |
-
2019
- 2019-08-01 CN CN201910707496.9A patent/CN110396501B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101050452A (en) * | 2007-03-27 | 2007-10-10 | 江苏省人民医院 | Method for separating stem cell of breast cancer |
| CN105274059A (en) * | 2015-06-15 | 2016-01-27 | 中山大学肿瘤防治中心 | Two breast cancer stem cell lines capable of being stably cultured and keeping original characteristics, separation method and uses thereof |
Non-Patent Citations (5)
| Title |
|---|
| Adrenomedullin is a key Protein Mediating Rotary Cell Culture System that Induces the Effects of Simulated Microgravity on Human Breast Cancer Cells;Li Chen 等;《Microgravity Sci. Technol》;20150610;第27卷;第417-426页 * |
| MammoCultTM Human Medium Kit;STEMCELL Technologies Inc.;《https://www.stemcell.com/products/mammocult-human-medium-kit.html#section-product-documents》;20190314;产品说明书第1页"产品描述"，第2页"MammoCultTM完全培养基的制备"，第2-3页"使用说明"步骤A-D * |
| MUC1-C Oncoprotein Activates ERK→C/EBPβ Signaling and Induction of Aldehyde Dehydrogenase 1A1 in Breast Cancer Cells;Maroof Alam 等;《JBC》;20131025;第288卷(第43期);摘要，第30894页左栏第3段，第30898页右栏至30899页左栏第1段，第30901页左栏 * |
| 乳腺癌干细胞富集与鉴定的初步研究;张颖 等;《现代肿瘤医学》;20190331;第27卷(第6期);摘要，第930页左栏第1段和第1.2节，第931页第2.4-2.5节，第933页左栏第1段 * |
| 应用旋转生物反应器培养小鼠胚胎干细胞的初步研究;魏国峰 等;《中国微生态学杂志》;20090630;第21卷(第6期);摘要，第527页右栏，第528页第1.2.2节，第529页第2.2节，第529-530页第3节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110396501A (en) | 2019-11-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110592022B (en) | Special culture medium for lung tumor organoid and 3D culture method without stent | |
| CN108504625B (en) | Mouse fibroblast and application thereof | |
| CN111411084A (en) | Culture medium and culture method for constructing liver tumor stent-free organoid | |
| CN113106065A (en) | Colorectal cancer organoid and liver organoid co-culture model and construction method thereof | |
| Low et al. | Neural precursor cells form rudimentary tissue-like structures in a rotating-wall vessel bioreactor | |
| CN115161283B (en) | Composition for directional differentiation and culture of liver portal bile duct cancer-derived organoids and application thereof | |
| CN105085938B (en) | The method that bletilla striata polyose water gelatin, culture matrix and its application are broken up with inducing umbilical cord mesenchymal stem to corneal epithelial cell | |
| CN110396501B (en) | Three-dimensional spheroid culture method for maintaining dryness of breast cancer stem cells in vitro | |
| CN107177551B (en) | Human intrahepatic bile duct cancer cell line with high tumor forming capability and application thereof | |
| CN108486039B (en) | Method for inducing human adipose-derived stem cells to differentiate into testicular interstitial cells by using small molecules | |
| CN112852709B (en) | Method for culturing mouse lung organoid | |
| CN108165529B (en) | Central memory T cell body and its in vitro culture method | |
| CN117230012A (en) | Human colon cancer organoid culture method and application thereof | |
| CN116478925A (en) | Method for quickly preparing tumor cell spheres in vitro | |
| CN117025505A (en) | Gastric mucosal epithelial precursor-like cell, and preparation method and application thereof | |
| CN108070560A (en) | A kind of isolation and culture method of the primary stomach cancer cell of people | |
| CN112608899B (en) | Application of serum-free culture medium in culturing spheroids of cancer tissue origin | |
| WO2022227289A1 (en) | Method for detecting sensitivity of organoid to macromolecular drug with sandwich culture system | |
| CN113106059B (en) | High-migration mesenchymal stem cells, and preparation method and application thereof | |
| CN114369573B (en) | Method for constructing in-situ primary nasopharyngeal carcinoma animal model | |
| CN109276712B (en) | Application of nano-synthetase in promoting synthesis of high-molecular hyaluronic acid in cells | |
| CN110628712B (en) | Preparation method and application of therapeutic interstage mesenchymal stem cells based on induced pluripotent stem cells | |
| CN114752626A (en) | Reversible immortalized II-type alveolar epithelial cell and construction and application thereof | |
| CN113322226A (en) | Exosome derived from three-dimensional cultured embryonic stem cells and application of exosome in preparation of medicine for treating hepatic fibrosis | |
| Xu et al. | 3D models of sarcomas: the next-generation tool for personalized medicine |
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 |