CN110408595A - A kind of cultural method of the tumour 3D organoid for gastric cancer test - Google Patents
A kind of cultural method of the tumour 3D organoid for gastric cancer test Download PDFInfo
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Abstract
The invention discloses a kind of cultural method of 3D tumour organoid (PDO) for gastric cancer anti-tumor drug sensitivity tests, be to solve existing tumour organoid modeling success rate it is low the problems such as.Specific step is as follows: step 1 by the present invention, prepares before testing；Step 2, sample of tissue；Step 3, tissue enzymatic hydrolysis separation；Step 4, the modeling of PDO tumour organoid；Step 5, PDO culture.The present invention has rational design, gastrin (Gastrin) is added during the cultivation process, to promote the modeling success rate and cell viability of gastric cancer organoid, it is formed by PDO agglomerate when the culture medium culture gastric cancer PDO of gastrin is added in discovery by comparison and significantly increases, cell viability also enhances, and modeling tumor formation rate also dramatically increases and reaches 80%, shows that using effect is good, can provide foundation for the test of tumour medicine sensibility, the screening of patient's anti-tumor drug and clinical application.
Description
Technical field
The present invention relates to gastric cancer testing field, the culture side of specifically a kind of tumour 3D organoid for gastric cancer test
Method.
Background technique
China so that global range in, gastric cancer be seriously threaten the most common malignant tumor of digestive tract of human health it
One, there is higher incidence and the death rate.Show that, in Quan Yazhou, the lethality of gastric cancer occupies malignant tumour the 2nd according to ASSOCIATE STATISTICS
Position.Most gastric cancers belong to gland cancer, early stage non-evident sympton, or the nonspecific symptoms such as epigastric discomfort, belch occur, often with
The stomaches chronic disease symptom such as gastritis, gastric ulcer is similar, is easily ignored, and therefore, the early diagnostic rate of gastric cancer is still lower, most
It has been middle and advanced stage when gastric cancer is diagnosed.Currently, the treatment of tumour still with operation based on, while be aided with radiotherapy and chemotherapy etc. with consolidate
Gu therapeutic effect, the method that additionally, there may be Chinese medicine treatment and biological therapy.Although industry is in Comprehensive Therapy on Gastric Carcinoma side in recent years
Face achieves considerable progress, but overall prognosis and pessimistic.
From the course of disease, the lethal reason of gastric cancer mainly includes drug resistance, recurrence and transfer.Wherein, tumour cell is to chemotherapy
Drug-resistant is one of the main reason for causing clinical treatment to fail.Traditional chemotherapy of gastric cancer drug is broadly divided into three categories: purple
China fir alcohols represents drug taxol (PTX)；Platinum class represents drugs Cisplatin (CDDP)；Fluorouracil represents drug 5- fluorine urine
Pyrimidine (5-Fu).These drugs have good inhibitory effect for the growth of malignant tumour at treatment initial stage, still, with treatment
Propulsion, malignant tumour gradually produces MDR to drug and (multidrug resistance: refers to when tumour cell is to a certain antitumor
When drug resistant, it to from not in contact with cross, structurally and functionally other entirely different anti-tumor drugs of mechanism can also be with
The phenomenon that generating drug resistance) so that the therapeutic effect of these front-line chemotherapeutic agents is bad.The generation of MDR has seriously affected pernicious
The therapeutic effect and post-operative recovery of tumour, cause Malignant Tumor Recurrence, influence prognosis.In addition for late gastric cancer patient, especially
It is the late gastric cancer patient of progression of disease after multi-thread treatment, needs to carry out the choosing of different therapeutic schemes according to the difference of the state of an illness
It selects.Due to the difference of individual inheritance background and the heterogeneity of tumour itself, different patients are also different for the reaction for the treatment of.
Therefore, a kind of good Indentification model how is established come the sensibility for judging tumour to tumour medicine, is in current curing gastric cancer
Pressing issues.
Foundation judges that tumour needs to collect many data to the sensibility system of tumour medicine, the tumour organoid of patient
Be exactly most true data, the cultural method of tumour organoid determines the characteristic of Neoplastic organ, existing tumour organoid into
The ingredient of culture medium includes: Advanced DMEM (improved DMEM culture medium) when row culture, B27, N2, GlutaMax,
Penicilin/streptomycin (penicillin/streptomycin), EGF (growth factor), Noggin, R-Spodin, FGF-10,
FGF-basic, Wnt-3A, Prostaglandin E2 (prostaglandin E2), Y-27632, Nicotinamide (niacinamide),
A83-01, SB202190, HGF (hepatocyte growth factor), this method although available tumour organoid, but obtain
The biomechanism of Neoplastic organ and the organ of patient itself have larger difference, cannot retain the pathomorphism of patient tissue, cannot
The behavior that primary tumor is simulated in the experiment in vitro of early stage can not be truly reflected tumorgenesis development, invasion, transfer etc.
The biological characteristics of aspect tumour, efficiency is relatively low when modeling, and only 20% or so success rate, this just influences what people obtained
The accuracy of judgement system.
Summary of the invention
A kind of cultural method for being designed to provide tumour 3D organoid for gastric cancer test of the embodiment of the present invention, with
Solve the problems mentioned above in the background art.
To achieve the above object, the embodiment of the present invention provides the following technical solutions:
A kind of cultural method of the tumour 3D organoid for gastric cancer test, the specific steps are as follows:
Step 1 prepares: the preparation before being tested before testing；
Step 2, sampling: obtaining clinical tumor tissue specimen, and tumor tissues sample carries out preliminary treatment and preservation, then
It is transported to laboratory；
Enzymatic hydrolysis separation: step 3 tumor tissues sample is washed using 10mL Advanced DMEM culture medium, to tumour
It is added in tissue specimen and contains mass fraction containing 10ml for 2%FCS (fetal calf serum, fetal calf serum) and 2mg/ml glue
The DMEM/F12 basal medium of former proteolytic enzyme, is then integrally placed on cyclotron oscillation device, handles 1- in 36-38 DEG C of constant temperature
2h, stop enzyme digestion reaction be centrifuged and be added 10ml containing mass fraction be 2%FCS improvement DMEM/F12 culture solution in be resuspended,
Obtain cell to be cultivated；
Step 4 configures culture medium: by the freeze thawing of Matrigel matrix and being mixed into homogenate shape, tissue culture plate is placed in
In ice bath, cell to be cultivated is mixed in tissue culture plate with Matrigel matrix and cell to be cultivated is suspended
In Matrigel matrix, then Matrigel matrix is added into tissue culture plate, then in 36-38 DEG C of placement 25-35min,
Obtain colloidal mixture；
Culture: Gastrin (gastrin) organoid culture medium, concentration 1nM- is added in step 5 in tissue culture plate
Tissue culture plate is placed in stationary culture in constant temperature and humidity incubator by 1000nM, the organoid culture medium of replacement in every 3-4 days,
It is passed within every 1-2 weeks, observes tumour 3D organoid culture form and growth rate with inverted microscope, choose growth speed
The highest system as tumour 3D organoid culture of rate, successfully pass on 3 times or more, the good sample of upgrowth situation can be with
Think that Kucheng's function is built in tumour 3D organoid culture, for long-term preservation sample, need to be carried out to the successful tumour 3D organoid in library is built
Freezen protective.
As further embodiment of the embodiment of the present invention: CO in incubator in step 52Volume fraction be 5%, temperature
It is 36-38 DEG C.
As further embodiment of the embodiment of the present invention: the preparation before testing in step 1 includes proposing Matrigel matrix
In the preceding refrigerator for being put into 4 DEG C from -20 DEG C for 24 hours, make its freeze thawing liquid condition；Advanced DMEM culture medium is set for 24 hours in advance
It is pre-chilled in centrifuge tube and in 4 DEG C of refrigerator.
As further embodiment of the embodiment of the present invention: it is tumor group that tumor tissues sample, which carries out preliminary treatment, in step 2
Sample is knitted to refrigerate at 4 DEG C.
As further embodiment of the embodiment of the present invention: the centrifugal acceleration being centrifuged in step 3 is 400g.
As further embodiment of the embodiment of the present invention: being mixed into Matrigel matrix using liquid transfer gun head in step 4
It is homogenized shape.
As further embodiment of the embodiment of the present invention: liquid transfer gun head be then placed in using preceding progress aseptic process it is sterile
In container, finally sterile chamber is placed in -20 DEG C of refrigerators and is pre-chilled.
As further embodiment of the embodiment of the present invention: tissue culture plate uses 24 well culture plates.
As further embodiment of the embodiment of the present invention: the concentration of Matrigel matrix is 150-200 μ L/ in step 4
cm2Grow area.
Compared with prior art, the beneficial effect of the embodiment of the present invention is:
The present invention has rational design, and gastrin is added during the cultivation process, with promote gastric cancer organoid modeling success rate and
Cell viability is formed by PDO agglomerate and obviously increased by comparison when the culture medium culture gastric cancer PDO of gastrin is added in discovery
Greatly, cell viability also enhances, and models tumor formation rate and also dramatically increase, and reaches 80%, shows that using effect is good, can be tumour
The test of drug susceptibility, the screening of patient's anti-tumor drug and clinical application provide foundation.
Detailed description of the invention
Fig. 1 is the tumour organoid form that comparative example obtains in the cultural method for the tumour 3D organoid tested for gastric cancer
Figure.
Fig. 2 is the tumour organoid shape that embodiment 1 obtains in the cultural method for the tumour 3D organoid tested for gastric cancer
State figure.
Fig. 3 is the tumour that embodiment 1 and comparative example obtain in the cultural method for the tumour 3D organoid tested for gastric cancer
The comparison diagram of organoid relative activity.
Fig. 4 is the tumour that embodiment 1 and comparative example obtain in the cultural method for the tumour 3D organoid tested for gastric cancer
The comparison diagram of organoid modeling success rate.
Specific embodiment
The technical solution of the patent is explained in further detail With reference to embodiment.
Embodiment 1
A kind of cultural method of the tumour 3D organoid for gastric cancer test, the specific steps are as follows:
Step 1 prepares before testing: Matrigel matrix is put into 4 from -20 DEG C for 24 hours in advance by the preparation before being tested
DEG C refrigerator in, make its freeze thawing liquid condition；Advanced DMEM culture medium is placed in centrifuge tube and 4 for 24 hours in advance
DEG C refrigerator in be pre-chilled, by liquid transfer gun head progress aseptic process be then placed in sterile chamber, sterile chamber is finally placed in -20
Pre-cooling in DEG C refrigerator；
Step 2, sampling: obtaining clinical tumor tissue specimen, and tumor tissues sample is in 4 DEG C of refrigerations and saves, and then transports
To laboratory；
Enzymatic hydrolysis separation: step 3 tumor tissues sample is washed using 10mL Advanced DMEM culture medium, to tumour
It is added in tissue specimen containing 10ml containing the basis DMEM/F12 that mass fraction is 2%FCS and 2mg/ml collagen hydrolase
Then culture medium is integrally placed on cyclotron oscillation device, handle 1.5h in 37 DEG C of constant temperature, stop enzyme digestion reaction and be centrifuged and be added
10ml obtains cell to be cultivated containing being resuspended in the improvement DMEM/F12 culture solution that mass fraction is 2%FCS；
Step 4 configures culture medium: by the freeze thawing of Matrigel matrix and being mixed into homogenate shape using liquid transfer gun head, will be thin
Born of the same parents' culture plate is placed in ice bath, and cell to be cultivated is mixed and made wait train in tissue culture plate with Matrigel matrix
Feeding cell is suspended in Matrigel matrix, then it is 180 μ L/cm that concentration, which is added, into tissue culture plate2Grow area
Matrigel matrix obtains colloidal mixture then in 37 DEG C of placement 30min；
Culture: step 5 Gastrin organoid culture medium is added in tissue culture plate, tissue culture plate is placed in perseverance
Stationary culture in constant temperature and humidity incubator, the organoid culture medium of replacement in every 3 days, is passed on every 2 weeks, micro- with being inverted
It is highest as tumour 3D organoid culture to choose growth rate for sem observation tumour 3D organoid culture form and growth rate
System, successfully pass on 3 times or more, the good sample of upgrowth situation can consider that Kucheng's function is built in tumour 3D organoid culture,
For long-term preservation sample, freezen protective need to be carried out to the successful tumour 3D organoid in library is built.
Comparative example
A kind of cultural method of the tumour 3D organoid for gastric cancer test, the specific steps are as follows:
Step 1 prepares: the preparation before being tested before testing；
Step 2, sampling: obtaining clinical tumor tissue specimen, and tumor tissues sample carries out preliminary treatment and preservation, then
It is transported to laboratory；
Enzymatic hydrolysis separation: step 3 tumor tissues sample is washed using 10mL Advanced DMEM culture medium, to tumour
It is added in tissue specimen containing 10ml containing the basis DMEM/F12 that mass fraction is 2%FCS and 2mg/ml collagen hydrolase
Then culture medium is integrally placed on cyclotron oscillation device, handle 1.5h in 37 DEG C of constant temperature, stop enzyme digestion reaction and be centrifuged and be added
10ml obtains cell to be cultivated containing being resuspended in the improvement DMEM/F12 culture solution that mass fraction is 2%FCS；
Step 4 configures culture medium: by the freeze thawing of Matrigel matrix and being mixed into homogenate shape, tissue culture plate is placed in
In ice bath, cell to be cultivated is mixed in tissue culture plate with Matrigel matrix and cell to be cultivated is suspended
In Matrigel matrix, then Matrigel matrix is added into tissue culture plate and obtains glue then in 37 DEG C of placement 30min
Shape mixture；
Culture: the organoid culture medium for not containing Gastrin is added, by cell culture in step 5 in tissue culture plate
Plate is placed in stationary culture in constant temperature and humidity incubator, and the organoid culture medium of replacement in every 4 days is passed on every 1 weeks, uses
Inverted microscope observes tumour 3D organoid culture form and growth rate, and it is highest as tumour 3D class to choose growth rate
The system of organ culture, successfully pass on 3 times or more, the good sample of upgrowth situation can consider that tumour 3D organoid culture is built
Kucheng's function need to carry out freezen protective to the successful tumour 3D organoid in library is built for long-term preservation sample.
The tumour organoid that embodiment 1 obtains and the tumour organoid that comparative example obtains are observed and tested, Fig. 1-4
As can be seen that the PDO agglomerate that the tumour organoid that embodiment 1 obtains is formed is significantly greater than the tumour organoid shape that comparative example obtains
At PDO agglomerate, the cell viability for the tumour organoid that embodiment 1 obtains is far longer than the tumour organoid that comparative example obtains
Cell viability, the modeling success rate for the tumour organoid that embodiment 1 obtains are 80%, and the tumour organoid that comparative example obtains is built
Mould success rate is 20%.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.No
It should treat any reference in the claims as limiting the claims involved.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.
Claims (13)
1. a kind of cultural method of the 3D tumour organoid for gastric cancer anti-tumor drug sensitivity tests, which is characterized in that tool
Steps are as follows for body:
Step 1 prepares before testing: tumour organoid culture medium and the tumour organoid culture medium containing Gastrin are prepared；
Sample of tissue: step 2 tumor tissues sample acquired during patients with gastric cancer operation or gastroscopic biopsy uses
Tissue washout liquid carries out primary wash, then carries out 4 DEG C of refrigerations using tissue preservation liquid, and turn tissue using tissue transport case
It is transported in Cell Culture Lab, tissue subsequent processing is carried out in Biohazard Safety Equipment；
Tissue enzymatic hydrolysis separation: step 3 tumor tissues sample is washed using 2mL tissue washout liquid three times, then in 37 DEG C of items
It under part, organizes enzymatic hydrolysis digestive juice to carry out tissue digestion 1 hour using 1ml, PBS is added after enzyme digestion reaction and carries out reaction terminating, it is low
Supernatant is abandoned after speed centrifugation, and is added in 1ml tumour organoid culture medium and be resuspended and cell count, is obtained to be cultivated thin
Born of the same parents；
Step 4,3D tumour organoid modeling: on 4 DEG C of ice chests by matrigel (Matrigel) melt and be mixed into homogenate
Cell to be cultivated is mixed in 1.5ml centrifuge tube with Matrigel and cell to be cultivated is suspended in by shape
It in Matrigel matrix, feeds the mixture into the tissue culture plate of pre-cooling, then in 36-38 DEG C of placement 30min, is coagulated
Solid colloidal mixture；
Tumour organoid culture: the organoid culture containing Gastrin is added on the upper layer of tissue culture plate matrigel in step 5
Tissue culture plate is placed in stationary culture in constant temperature and humidity incubator by base, and every 3-4 days one subcultures of replacement carry out for every 1-2 weeks
Passage.
2. the culture side of the 3D tumour organoid according to claim 1 for gastric cancer anti-tumor drug sensitivity tests
Method, which is characterized in that the tumour organoid culture medium of the step 1 contains following ingredient: Advanced DMEM is (improved
DMEM culture medium), B27, N2, GlutaMax, Penicilin/streptomycin (penicillin/streptomycin), (epidermis is raw by EGF
The long factor), Noggin (noggin), R-Spodin, FGF-10 (Fibroblast growth factor 1 0), FGF-basic (basic fibroblast
Growth factor), Wnt-3A, Prostaglandin E2 (prostaglandin E2), Y-27632, Nicotinamide (niacinamide),
A83-01, SB202190, HGF (hepatocyte growth factor).
3. the culture side of the 3D tumour organoid according to claim 1 for gastric cancer anti-tumor drug sensitivity tests
Method, which is characterized in that the tumour organoid culture medium containing Gastrin of the step 2 is the base in tumour organoid culture medium
The Gastrin of 10nM is added on plinth.
4. the culture side of the 3D tumour organoid according to claim 1 for gastric cancer anti-tumor drug sensitivity tests
Method, which is characterized in that the tissue washout liquid of the step 2 is to contain 2% dual anti-(penicillin/streptomycin) phosphate buffer
(PBS)。
5. the culture side of the 3D tumour organoid according to claim 1 for gastric cancer anti-tumor drug sensitivity tests
Method, which is characterized in that the tissue preservation liquid of the step 2 is the DMEM culture medium dual anti-containing 3% fetal calf serum and 2%.
6. the culture side of the 3D tumour organoid according to claim 1 for gastric cancer anti-tumor drug sensitivity tests
Method, which is characterized in that the tissue transport case of the step 2 is the foam thermal insulation box for adding ice cube, has heat preservation and anticollision energy
Power.
7. the culture side of the 3D tumour organoid according to claim 1 for gastric cancer anti-tumor drug sensitivity tests
Method, which is characterized in that be the desinfection chamber that clean rank is ten thousand grades between the cell culture of the step 2.
8. the culture side of the 3D tumour organoid according to claim 1 for gastric cancer anti-tumor drug sensitivity tests
Method, which is characterized in that the tissue enzymatic hydrolysis digestive juice of the step 3 has action temperature and the feature small to cellular damage.
9. the culture side of the 3D tumour organoid according to claim 1 for gastric cancer anti-tumor drug sensitivity tests
Method, which is characterized in that the matrigel of the step 4 needs the preservation at -20 DEG C of temperature below, and matrigel is solid at this time
State；Melt under the conditions of 4 DEG C is liquid；Under the conditions of 37 DEG C, after placing 30min, it is solid that matrigel will solidify agglutination
Body.
10. the culture side of the 3D tumour organoid according to claim 1 for gastric cancer anti-tumor drug sensitivity tests
Method, which is characterized in that CO in the incubator of the step 52Volume fraction be 5%, temperature be 37 DEG C.
11. the culture side of the 3D tumour organoid according to claim 1 for gastric cancer anti-tumor drug sensitivity tests
Method, it is characterised in that: the centrifugal acceleration being centrifuged in the step 3 is 400-1000g.
12. the culture side of the 3D tumour organoid according to claim 1 for gastric cancer anti-tumor drug sensitivity tests
Method, it is characterised in that: the tissue culture plate is using 24 well culture plates or 96 well culture plates or 12 well culture plates.
13. the culture side of the 3D tumour organoid according to claim 3 for gastric cancer anti-tumor drug sensitivity tests
Method, it is characterised in that: the use concentration range of Gastrin is 1nM-100nM.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113897336A (en) * | 2020-06-22 | 2022-01-07 | 厦门博创盛世生物技术有限公司 | Preparation method and application of WRN conditioned medium |
| CN114908039A (en) * | 2022-06-17 | 2022-08-16 | 浙江弘瑞医疗科技有限公司 | Culture medium for stomach cancer organoid and culture method without bracket thereof |
| CN115094041A (en) * | 2022-08-25 | 2022-09-23 | 杭州艾名医学科技有限公司 | Stomach cancer organoid culture medium and culture method |
| CN115369090A (en) * | 2022-09-05 | 2022-11-22 | 上海中医药大学马鞍山康复研究院 | Method for preparing homologous stomach cancer organoid |
| CN116590235A (en) * | 2023-07-14 | 2023-08-15 | 北京嘉士腾医学检验实验室有限公司 | Efficient digestive tract tumor organoid culture method |
| CN116590377A (en) * | 2023-07-17 | 2023-08-15 | 安泰康生物技术(北京)有限公司 | Method for detecting sensitivity of patient-derived organ type tissue culture medicament |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108070560A (en) * | 2016-11-15 | 2018-05-25 | 江苏齐氏生物科技有限公司 | A kind of isolation and culture method of the primary stomach cancer cell of people |
| CN109136188A (en) * | 2017-06-15 | 2019-01-04 | 上海集技生物技术有限公司 | A kind of culture of biopsy intestinal canal tumour organoid is passed on, is frozen and method for resuscitation and its application |
| CN109136163A (en) * | 2011-06-10 | 2019-01-04 | 荷兰皇家科学院 | Culture medium for stem cell |
| CN109628402A (en) * | 2019-02-13 | 2019-04-16 | 妙顺（上海）生物科技有限公司 | A kind of primary organoid cultural method of gastric cancer tumor |
-
2019
- 2019-08-23 CN CN201910783626.7A patent/CN110408595A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109136163A (en) * | 2011-06-10 | 2019-01-04 | 荷兰皇家科学院 | Culture medium for stem cell |
| CN108070560A (en) * | 2016-11-15 | 2018-05-25 | 江苏齐氏生物科技有限公司 | A kind of isolation and culture method of the primary stomach cancer cell of people |
| CN109136188A (en) * | 2017-06-15 | 2019-01-04 | 上海集技生物技术有限公司 | A kind of culture of biopsy intestinal canal tumour organoid is passed on, is frozen and method for resuscitation and its application |
| CN109628402A (en) * | 2019-02-13 | 2019-04-16 | 妙顺（上海）生物科技有限公司 | A kind of primary organoid cultural method of gastric cancer tumor |
Non-Patent Citations (3)
| Title |
|---|
| MIRANDA LIN等: "Utilizing gastric cancer organoids to assess tumor biology and personalize medicine", 《WORLD JOURNAL OF GASTROINTESTINAL ONCOLOGY》 * |
| SINA BARTFELD等: "In Vitro Expansion of Human Gastric Epithelial Stem Cells and Their Responses to Bacterial Infection", 《GASTROENTEROLOGY》 * |
| THERESE SEIDLITZ等: "Human gastric cancer modelling using organoids", 《GUT》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113897336A (en) * | 2020-06-22 | 2022-01-07 | 厦门博创盛世生物技术有限公司 | Preparation method and application of WRN conditioned medium |
| CN114908039A (en) * | 2022-06-17 | 2022-08-16 | 浙江弘瑞医疗科技有限公司 | Culture medium for stomach cancer organoid and culture method without bracket thereof |
| CN115094041A (en) * | 2022-08-25 | 2022-09-23 | 杭州艾名医学科技有限公司 | Stomach cancer organoid culture medium and culture method |
| CN115369090A (en) * | 2022-09-05 | 2022-11-22 | 上海中医药大学马鞍山康复研究院 | Method for preparing homologous stomach cancer organoid |
| CN116590235A (en) * | 2023-07-14 | 2023-08-15 | 北京嘉士腾医学检验实验室有限公司 | Efficient digestive tract tumor organoid culture method |
| CN116590235B (en) * | 2023-07-14 | 2023-11-21 | 北京嘉士腾医学检验实验室有限公司 | Efficient digestive tract tumor organoid culture method |
| CN116590377A (en) * | 2023-07-17 | 2023-08-15 | 安泰康生物技术(北京)有限公司 | Method for detecting sensitivity of patient-derived organ type tissue culture medicament |
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