CN109136188A - A kind of culture of biopsy intestinal canal tumour organoid is passed on, is frozen and method for resuscitation and its application - Google Patents
A kind of culture of biopsy intestinal canal tumour organoid is passed on, is frozen and method for resuscitation and its application Download PDFInfo
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- CN109136188A CN109136188A CN201710483803.0A CN201710483803A CN109136188A CN 109136188 A CN109136188 A CN 109136188A CN 201710483803 A CN201710483803 A CN 201710483803A CN 109136188 A CN109136188 A CN 109136188A
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- 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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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- 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
Abstract
The present invention relates to a kind of cultures of biopsy intestinal canal tumour organoid, passage, it freezes and method for resuscitation and its application method, by the way that a small amount of biopsy tumor tissues are cultivated and are passed on, it can turn out consistent with its source cancerous tissue genetic background height, its genetic mutation and the matched organoid of discontinuity height in corresponding tissue specimen, the organoid of culture can also freeze and recover simultaneously, culture and efficacy testing time are shorter, toxigenic capacity is compared with PDTX model economy, establishing tumour organoid sample storehouse may be implemented high flux screening drug, the organoid of culture can be with test sample radiation sensitive etc., it is widely used.
Description
Technical field
The present invention relates to the method for tumour organoid culture, the culture of specifically a kind of biopsy intestinal canal tumour organoid is passed
In generation, freezes and method for resuscitation and its application.
Background technique
In tumor research, the PDTX model especially in the screening of newtype drug, the evaluation studies of individualized treatment
(people source tumor xenograft is in immunodeficient mouse model) obtains certain effect, and transplantation tumor also remains Primary Tumor
Differentiation degree, morphological feature, design feature and molecular biology fundamental characteristics and tumor microenvironment.But this model
Also in place of the defect for having it, although PDTX model simulates the biological characteristics of Primary Tumor, can provide body for the research of tumour
Interior simulated environment, but the foundation of this model is not completely identical with Primary Tumor after all, and tumor formation model time is long, is established
Model relative difficulty, immunodeficient animals are at high cost, while there is also certain ethics problems.PDTX model, in liver cancer, pancreas
Gland cancer, prostate cancer, breast cancer and glioma research in have been reported that.But it will be seen that this tumour transplatation
The foundation of model is relatively difficult, especially some knub position such as tumor in digestive tract, because its original position is in alimentary canal chamber
Interior, the survival rate and success rate of transplanting are all very low, therefore hinder its extensive use, are only limited to certain special swollen
Tumor.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of culture of biopsy intestinal canal tumour organoid, passes on, freezes
With method for resuscitation and its application, to overcome defect of the existing technology.
The technical scheme to solve the above technical problems is that
A kind of cultural method of biopsy intestinal canal tumour organoid, includes the following steps:
1) the biopsy tumor tissues for taking the grain of rice to soya bean size are placed in ice PBS (penicillin 100IU/ml+
100 μ g/ml of streptomycin) in buffer；
2) using PBS (100 μ g/ml of the penicillin100IU/ml+streptomycin) buffering for being pre-chilled 5-10 minutes
Liquid repeatedly rinses biopsy tumor tissues well；
3) sterile chopping tumor tissues and move into be preheated in 37 DEG C of enzymic digestion liquid, concussion digestion 30-60 minutes；
4) it is blown and beaten repeatedly using liquid-transfering gun until tumor fragment disappears, and is centrifuged with 100g-300g revolving speed after digesting
5 minutes；
5) supernatant is removed, is centrifuged repeatedly within 60gx 5 minutes repeatedly；
6) supernatant is removed, adds 10ml pre-cooling PBS (100 μ g/ml of penicillin 100IU/ml+streptomycin) slow
Fliud flushing takes 10ul microscopically observation to count crypts quantity, is centrifuged within 100-300g 5 minutes, clean supernatant is removed, with 100-300
A crypts/50ul Matrigel ratio is resuspended 3D and cultivates organoid.
A kind of propagating method of biopsy intestinal canal tumour organoid is also provided, is included the following steps:
1) the primary organoid prepared according to the above method is taken, culture medium is removed, goes for 1ml PBS buffer solution for cleaning one time
Except liquid, 1ml ice PBS buffer solution is rejoined, Matrigel is smashed repeatedly with pipette tips, moves into 50ml centrifuge tube；
2) 100g-300g is centrifuged 5 minutes, removes supernatant, and PBS buffer solution on the rocks is resuspended, blows and beats repeatedly, this step repeats 3-
Exist to no gel for 5 times, organoid uniformly smashed；
3) after last time is centrifuged, clean PBS buffer solution is gone, Matrigel is resuspended, and is inoculated with and trains according to 1: 3-1: 5 ratios
It supports.
Also provide a kind of biopsy intestinal canal tumour organoid freeze and method for resuscitation, which comprises the steps of:
1) freeze: passage organoid culture 3-5 days after, according to propagating method step 1) -2) in cleaning way clean to
For the last time, frozen stock solution made of 95%FBS+5%DMSO is added after removing PBS buffer solution, freezing storing box is put into -80 DEG C of ice
Case moves into liquid nitrogen storage after 16 hours；
2) it thaws: freezing organoid and be removed from liquid nitrogen rear 37 DEG C of water-baths and thaw rapidly and move into the DMEM containing 10%FBS
In, 100-300g is centrifuged to obtain precipitating, carries out inoculated and cultured by above-mentioned cultural method.
Finally, additionally providing a kind of application of biopsy intestinal canal tumour organoid, it is used for chemotherapeutics test, steps are as follows
Organoid passes on different algebra and is inoculated with 48 orifice plates with 100-200 organoid/25 μ l, and after 24 hours plus various concentration 5- fluorine is urinated
Pyrimidine (5-FU) (1 μm/L, 10 μm/L, 100 μm/L, 300 μm/L) or Irinotecan (CPT-11) (0.1 μm/L, 1 μm/L,
10 μm/L, 100 μm/L) chemotherapeutics, culture solution is replaced every three days, and Survival clone and the test survival of cck8 method are shot after 6 days
Rate.It is used for radiation sensitive test, and steps are as follows: organoid passage, different algebra are connect with 100-200 organoid/25 μ l
48 orifice plates of kind, x-ray various dose (0Gy, 3Gy, 6Gy, 9Gy, 12Gy and 15Gy) is irradiated after 24 hours, every three days replacement culture
Liquid, shoots Survival clone after 6 days and cck8 method tests survival rate.
The beneficial effects of the present invention are: by the way that a small amount of biopsy tumor tissues are cultivated and are passed on, can turn out with
Its source cancerous tissue genetic background height is consistent, genetic mutation and the matched class device of the discontinuity height in corresponding tissue specimen
Official, while the organoid cultivated can also freeze and recover, culture and efficacy testing time are shorter, and toxigenic capacity is compared with PDTX model
Economy, establishing tumour organoid sample storehouse may be implemented high flux screening drug, and the organoid of culture can be with test sample ray
Sensibility etc. is widely used.
Specific embodiment
The principles and features of the present invention are described below, and the given examples are served only to explain the present invention, is not intended to limit
Determine the scope of the present invention.
One, the cultural method of biopsy intestinal canal tumour organoid
1. the biopsy tumor tissues of the grain of rice to soya bean size are placed in ice PBS (penicillin 100IU/ml+
streptomycin 100μg/ml)。
2. it is more that PBS (100 μ g/ml of penicillin 100IU/ml+streptomycin) cleaning in 5-10 minutes is pre-chilled
It is secondary, according to sample clean level.
3. sterile chopping tumor tissues move into preheating enzymic digestion liquid, 37 DEG C of water-baths digest 30-60 minutes.
4. liquid-transfering gun is blown and beaten repeatedly until tumor fragment disappears after digestion, 100g-300g is centrifuged 5 minutes.
5. removing supernatant, it is centrifuged repeatedly within 60gx 5 minutes repeatedly.
6. removing supernatant, add 10ml that PBS is pre-chilled, 10 μ l microscopically observations is taken to count crypts quantity, 100-300g 5 divides
Zhongli's heart, removes clean supernatant, and 3D culture organoid (100-300 crypts/50 μ l Matrigel) is resuspended in Matrigel.
Two, the passage of organoid
1. primary crypts culture 5-10 days, removing culture medium, 1mlPBS cleans a removal liquid, rejoins 1ml ice
PBS smashes Matrigel with pipette tips repeatedly, moves into 50ml centrifuge tube.
2.100g-300g is centrifuged 5 minutes, carefully removes supernatant, and PBS on the rocks is resuspended, blows and beats repeatedly, this step repeats 3-5
Secondary to exist to no gel, while organoid is uniformly smashed.
3. after last time is centrifuged, going clean PBS, Matrigel to be resuspended, 1: 3-1: 5 inoculated and cultureds.
Three, organoid freezes and recovers
1. after passage organoid culture 3-5 days, cleaning by propagating method to last time, appropriate freeze is added after removing PBS
Liquid storage (95%FBS+5%DMSO), freezing storing box are put into -80 DEG C of refrigerators, and liquid nitrogen storage is moved into after 16 hours.
It thaws rapidly and moves into the DMEM containing 10%FBS 2. freezing organoid and being removed from liquid nitrogen rear 37 DEG C of water-baths,
100-300g is centrifuged to obtain precipitating, by primary vaccination ways inoculated and cultured.
Four, the chemotherapeutics test of organoid
Organoid passage, different algebra are inoculated with 48 orifice plates with 100-200 organoid/25 μ l, after 24 hours plus different dense
Spend 5-FU (1 μm/L, 10 μm/L, 100 μm/L, 300 μm/L) or CPT-11 (0.1 μm/L, 1 μm/L, 10 μm/L, 100 μm/L)
Equal chemotherapeutics, replace culture solution every three days, and Survival clone is shot after 6 days and cck8 method tests survival rate.
Five, the radiation-sensitive test of organoid
Organoid passage, different algebra are inoculated with 48 orifice plates with 100-200 organoid/25 μ l, and x-ray is different after 24 hours
Dosage (0Gy, 3Gy, 6Gy, 9Gy, 12Gy and 15Gy) irradiation, replaces culture solution every three days, and Survival clone and cck8 are shot after 6 days
Method tests survival rate.
Digestive ferment: 1%FBS DMEM (Gibco) contains
Collagenase IV (Sigma), 300-600IU/ml；
Ly27632 (Sigma), 5-20 μM
Culture solution: Advanced DMEM/F12 medium (Gibco) contains
1x N2(Invitrogen)；
1X B27(Invitrogen)；
Nicotinamide (Sigma), 5-20mM；
Human EGF (Peprotech), 50-100ng/ml；
Gastrin (Sigma), 10-30nM；
A83-01 (Sigma), 300-500nM；
SB202190 (Sigma), 3-10 μM；
Prostaglandin E2 (Peprotech), 10-50nM；
Normocin (Invitrogen), 100-300mg/ml；
Glutamax (Gibco), 10-50mM；
HEPES (Gibco), 10-50mM；
N-acetylcysteine (Sigma), 1-5 μM；
R-spondin 1 (Peprotech), 500-1000ng/ml；
Noggin (Peprotech), 100-500ng/ml；
The present invention has the advantage that
1. a small amount of biopsy tumor tissues may conform to extract and require.
2. enteron aisle cancerous tissue can very easily turn out tumour organoid after digesting.
3. the intestinal canal tumour organoid genetic background of culture is consistent with its source cancerous tissue genetic background height, heredity is prominent
Become and is matched with the discontinuity height in corresponding tissue specimen.
4. the intestinal canal tumour organoid of culture can freeze and recover.
5. culture and efficacy testing time are shorter, toxigenic capacity is compared with PDTX model economy.
6. same sample organoid can test not same drug, PDTX model subjects difference is not present.
7. establishing tumour organoid sample storehouse may be implemented high flux screening drug.
8. the organoid of culture can be with test sample radiation sensitive.
9. ethics problem is not present.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (6)
1. a kind of cultural method of biopsy intestinal canal tumour organoid, which comprises the steps of:
1) the biopsy tumor tissues for taking the grain of rice to soya bean size are placed in ice PBS buffer solution；
2) biopsy tumor tissues are repeatedly rinsed well using the PBS buffer solution for being pre-chilled 5-10 minutes；
3) sterile chopping tumor tissues and move into be preheated in 37 DEG C of enzymic digestion liquid, concussion digestion 30-60 minutes；
4) it is blown and beaten repeatedly using liquid-transfering gun until tumor fragment disappears, and is centrifuged 5 points with 100g-300g revolving speed after digesting
Clock；
5) supernatant is removed, is centrifuged repeatedly within 60gx5 minutes repeatedly；
6) supernatant is removed, adds 10m1 that PBS buffer solution is pre-chilled, 10 μ l microscopically observations is taken to count crypts quantity, 100-300g5 divides
Zhongli's heart removes clean supernatant, and 3D is resuspended with 100-300 crypts/50 μ l Matrigel ratios and cultivates organoid.
2. a kind of cultural method of biopsy intestinal canal tumour organoid according to claim 1, it is characterised in that: the PBS
Buffer is that 100 μ g/ml of penicillin 100IU/ml+streptomycin is mixed, and enzymic digestion liquid is 1%FBS
DMEM+Collagenase IV300-600IU/ml+Ly27632 5-20 μM is mixed.
3. a kind of propagating method of biopsy intestinal canal tumour organoid, which comprises the steps of:
1) the primary organoid for taking method preparation according to claim 1, removes culture medium, and 1ml PBS buffer solution is cleaned one time and gone
Except liquid, 1ml ice PBS buffer solution is rejoined, Matrigel is smashed repeatedly with pipette tips, moves into 50ml centrifuge tube；
2) 100g-300g is centrifuged 5 minutes, removes supernatant, and PBS buffer solution on the rocks is resuspended, blows and beats repeatedly, this step repeats 3-5 times
Exist to no gel, organoid is uniformly smashed；
3) after last time is centrifuged, clean PBS buffer solution is gone, Matrigel is resuspended, according to 1: 3-1: 5 ratio inoculated and cultureds.
4. a kind of biopsy intestinal canal tumour organoid freeze and method for resuscitation, which comprises the steps of:
1) freezing: after passage organoid culture 3-5 days, step 1) -2 according to claim 2) cleaning method cleans to most
It is primary afterwards, it removes and frozen stock solution made of 95%FBS+5%DMSO is added after PBS buffer solution is put into freezing storing box, freezing storing box is put into-
80 degree of refrigerators move into liquid nitrogen storage after 16 hours；
2) it thaws: freezing organoid and be removed from liquid nitrogen rear 37 DEG C of water-baths and thaw rapidly and move into the DMEM containing 10%FBS,
100-300g is centrifuged to obtain precipitating, carries out inoculated and cultured by claim 1 the method.
5. a kind of application of biopsy intestinal canal tumour organoid is used for chemotherapeutics test, it is characterised in that: organoid passage,
Different algebra are inoculated with 48 orifice plates with 100-200 organoid/25 μ l, and after 24 hours plus concentration is respectively 1 μm/L, 10 μm/L, 100
μm/L, the 5 FU 5 fluorouracil of 300 μm/L or 0.1 μm/L, 1 μm/L, 10 μm/L, the Irinotecan chemotherapeutics of 100 μm/L, often
Three days replacement culture solutions, shoot Survival clone after 6 days and cck8 method tests survival rate.
6. a kind of application of biopsy intestinal canal tumour organoid is used for radiation sensitive test, it is characterised in that: organoid passes
Generation, different algebra are inoculated with 48 orifice plates with 100-200 organoid/25 μ l, x-ray various dose 0Gy, 3Gy after 24 hours, 6Gy,
9Gy, 12Gy and 15Gy irradiation, replace culture solution every three days, and Survival clone is shot after 6 days and cck8 method tests survival rate.
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| CN110408595A (en) * | 2019-08-23 | 2019-11-05 | 厦门博创盛世生物技术有限公司 | A kind of cultural method of the tumour 3D organoid for gastric cancer test |
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| CN112725279A (en) * | 2020-12-31 | 2021-04-30 | 纳肽得(青岛)生物医药有限公司 | Drug sensitivity detection and standard establishment method based on tumor organoid model and application of microfluidic chip structure |
| CN113287599A (en) * | 2021-04-30 | 2021-08-24 | 创芯国际生物科技(广州)有限公司 | Cryopreservation liquid composition, cryopreservation liquid and organoid cryopreservation method |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110408595A (en) * | 2019-08-23 | 2019-11-05 | 厦门博创盛世生物技术有限公司 | A kind of cultural method of the tumour 3D organoid for gastric cancer test |
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| CN112725279A (en) * | 2020-12-31 | 2021-04-30 | 纳肽得(青岛)生物医药有限公司 | Drug sensitivity detection and standard establishment method based on tumor organoid model and application of microfluidic chip structure |
| CN112725279B (en) * | 2020-12-31 | 2023-10-24 | 纳肽得(青岛)生物医药有限公司 | Drug sensitivity detection and standard establishment method based on tumor organoid model and application of microfluidic chip structure |
| CN113287599A (en) * | 2021-04-30 | 2021-08-24 | 创芯国际生物科技(广州)有限公司 | Cryopreservation liquid composition, cryopreservation liquid and organoid cryopreservation method |
| CN113287599B (en) * | 2021-04-30 | 2022-04-19 | 创芯国际生物科技(广州)有限公司 | Cryopreservation liquid composition, cryopreservation liquid and organoid cryopreservation method |
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| CN114107388A (en) * | 2021-11-12 | 2022-03-01 | 复旦大学附属中山医院 | Method for transfecting organoid by adenovirus |
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