CN109628402A - A kind of primary organoid cultural method of gastric cancer tumor - Google Patents
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Abstract
The present invention relates to cytobiology technologies and application field, and in particular to a kind of primary organoid cultural method of gastric cancer tumor.This method step are as follows: feeder cells, gastric cancer tumor primary cell are obtained using enzyme digestion-cell culture method respectively；To the feeder cells, gastric cancer tumor primary cell, it is separately added into magnetic suspension particle, obtains feeder cells, gastric cancer tumor primary cell containing magnetic particle through incubation-digestion；Feeder cells containing magnetic particle, the gastric cancer tumor primary cell containing magnetic particle are seeded to the primary organoid culture medium of gastric cancer tumor, cultivated under the conditions of magnetic drive, the primary organoid of gastric cancer tumor is obtained.Feeder cells and gastric cancer tumor primary cell are co-cultured by using magnetic suspension culture systems, more preferable simulation tumor microenvironment and intercellular interaction provide new pharmacy in vitro test method for medicament research and development industry.
Description
Technical field
The present invention relates to cytobiology technologies and application field, and in particular to a kind of primary organoid culture of gastric cancer tumor
Method.
Background technique
Gastric cancer is the common malignant tumour in the whole world, and prognosis is relatively poor, seriously threatens human health.At present in drug research
During, pharmacy in vitro test is typically considered the important means of testing of evaluation drug effectiveness.
In the prior art, main that the drug developments mistakes such as pharmacy in vitro test are carried out using two-dimentional tumour primary culture method
Journey, for example tumor cell suspension is obtained first, using RPMI1640 culture medium and basic fibroblast growth factor
(basic fiibroblast growth factor, bFGF), 20ng/mL epidermal growth factor (epithelial growth
Factor, EGF), bovine serum albumin(BSA) (the bovine serum of 5 μ g/mL insulin (insulin) and 0.4g/100mL
Albumin, BSA) etc. growth factors to cell carry out separation with cultivate acquisition Tumor cell, this method can be improved tumour
The success rate of primary cultured cell, and can be reduced interference of the Tumor cell by non-tumor cell.
But the Tumor cell that the above method obtains can simulate tumour migration in vivo and invasion shape well
Condition has significant limitation in terms of evaluating anti-tumor drug validity.
Summary of the invention
For the deficiencies in the prior art, the present invention provides a kind of primary organoid cultural methods of gastric cancer tumor.
Specific steps are as follows:
Feeder cells, gastric cancer tumor primary cell are obtained using enzyme digestion-cell culture method respectively；
The feeder cells, the gastric cancer tumor primary cell that culture density is 75%-85% are chosen, magnetcisuspension is separately added into
Floating particle is uniformly mixed and is placed in incubation 10-12h in incubator；
Feeder cells, gastric cancer tumor primary cell after taking the incubation, be separately added into pancreas enzyme -EDTA-digestive juice into
Cell culture medium is added after row digestion to mix, and it is former to be centrifuged feeder cells, gastric cancer tumor of the 4-8min acquisition containing magnetic particle
For cell；
The feeder cells containing magnetic particle, gastric cancer tumor primary cell are seeded to the primary organoid of gastric cancer tumor
Culture medium is cultivated 15-25 days under the conditions of magnetic drive, obtains the primary organoid of gastric cancer tumor.
Technical solution provided by the invention include it is following the utility model has the advantages that
Feeder cells and gastric cancer tumor primary cell are co-cultured by using magnetic suspension culture systems, preferably
Tumor microenvironment and intercellular interaction are simulated, new pharmacy in vitro test method is provided for medicament research and development industry,
And this method has many advantages, such as that easy to operate, cost is relatively low.
Detailed description of the invention
Attached drawing 1 is 3D co-culture model schematic device；
Attached drawing 2 is the morphosis of feeder cells；
Attached drawing 3 is the morphosis of gastric cancer tumor primary cell；
Attached drawing 4 is the morphosis of the primary organoid of gastric cancer tumor.
Specific embodiment
In order to more fairly set out technology contents of the invention, it is described in detail in conjunction with specific embodiments herein, it is clear that
Cited embodiment is the preferred embodiment of the technical program, and those skilled in the art can be according to disclosed skill
The other technologies scheme that art content is apparent from still falls within protection scope of the present invention.
Embodiment:
Feeder cells, gastric cancer tumor primary cell are obtained using enzyme digestion-cell culture method respectively.
The method for obtaining feeder cells, specifically includes the following steps:
The postoperative isolated Tumor-surrounding tissue of solid tumor patient is weighed and recorded (4.3g), is immersed in equipped with 20ml Hank ' s
In the 50ml centrifuge tube of balanced salt solution, it is transferred to laboratory in the shortest time, and strictly carries out sterile working；
70% alcohol disinfecting is sprayed to the 50ml centrifugation pipe surface equipped with Tumor-surrounding tissue, 50ml centrifuge tube is moved into biology peace
In full cabinet, extra alcohol is removed, opens centrifuge tube pipe lid, Tumor-surrounding tissue is moved into 100mm culture dish using sterile blunt nosed tweezer
In, clot is removed, uses Hank ' s balanced salt solution rinse Tumor-surrounding tissue three times, keeps Tumor-surrounding tissue wet；
Paste is become using sterile scissors chopping Tumor-surrounding tissue, is moved it into 50ml centrifuge tube, according to every 1g cancer
0.1% II Collagenase Type 15.05ml is added in the ratio in week tissue 3.5ml, covers tightly centrifuge tube pipe lid；
Centrifuge tube is put into 37 C water baths to be incubated for 40 minutes, avoids cell mass heavy every the 5 minutes light centrifuge tube that shakes
Bottom；Centrifuge tube is put into the centrifuge of 4 degrees Celsius of pre-coolings, 300g is centrifuged 3 minutes, carefully removes supernatant with pipette；
15ml feeder cells culture medium (the DMEM culture medium containing 10% people's AB serum) is added into centrifuge tube, with shifting
Liquid pipe gently blows and beats resuspension to cell mass, crosses 100um strainer, collects filtrate；
Filtrate is moved into a new 50ml centrifuge tube and covers tightly centrifuge tube pipe lid, 300g is centrifuged 3 minutes, is preheated using 5ml
Feeder cells culture medium cell is resuspended, and to cell count, with 1*105The density of a/ml is seeded in T75 culture bottle,
Every 2 days replacement fresh feeder confluent monolayer cells culture mediums of follow-up cultivation obtain feeder cells.
The method for obtaining gastric cancer tumor primary cell, specifically includes the following steps:
The postoperative isolated gastric cancer tumor tissue of solid tumor patient is weighed and recorded (3.55g), is immersed in equipped with 20ml
In the 50ml centrifuge tube of Hank ' s balanced salt solution, it is transferred to laboratory in the shortest time, and strictly carries out sterile working；
70% alcohol disinfecting is sprayed to the 50ml centrifugation pipe surface equipped with gastric cancer tumor tissue, 50ml centrifuge tube is moved into and is given birth to
In object safety cabinet, extra alcohol is removed, centrifuge tube pipe lid is opened, is moved into gastric cancer tumor tissue using sterile blunt nosed tweezer
In 100mm culture dish, clot is removed, uses Hank ' s balanced salt solution rinse Tumor-surrounding tissue three times, keeps gastric cancer tumor tissue
It is wet；
Paste is become using sterile scissors chopping gastric cancer tumor tissue, is moved it into 50ml centrifuge tube, according to every
The digestive juice 17.75ml of the IV Collagenase Type containing 0.1% is added in the ratio of 1g gastric cancer tumor tissue 5ml, covers tightly centrifuge tube pipe
Lid；
Centrifuge tube is put into 37 C water baths to be incubated for 120 minutes, avoids cell mass heavy every the 5 minutes light centrifuge tube that shakes
Bottom；Centrifuge tube is put into the centrifuge of 4 degrees Celsius of pre-coolings, 300g is centrifuged 3 minutes, carefully removes supernatant with pipette；
15ml feeder cells culture medium (the DMEM culture medium containing 10% people's AB serum) is added into centrifuge tube, with shifting
Liquid pipe gently blows and beats resuspension to cell mass, crosses 100um strainer, collects filtrate；
Filtrate is moved into a new 50ml centrifuge tube and covers tightly centrifuge tube pipe lid, 300g is centrifuged 3 minutes, is pre-chilled using 5ml
Feeder cells culture medium cell is resuspended, and to cell count, coating someone in advance is seeded to the density of 3*106/ml
In the T75 culture bottle of fibronectin.
Culture medium is directly sucked out from culture bottle into centrifuge tube after culture 30 minutes, 300g is centrifuged 3 minutes, is discarded
Clearly, (contain 10%noggin, 50%Wnt-3a, 1nM gastrin, 10%ITS- using gastric cancer tumor primitive cell culture base
A, the DMEM/F12 culture medium of 1%N2,2%B-27,100ng/ml EGF, 200ng/ml FGF) it is resuspended, with 7*105A/
The density of ml is seeded to, and renews within subsequent every 3 days half fresh gastric cancer tumor primitive cell culture base, obtains gastric cancer tumor primary cell.
Magnetic suspension particle is removed from refrigerator, and in thaw at RT 15min.Choose the raising that culture density is about 80%
Magnetic suspension particle is added into culture bottle or culture dish for confluent monolayer cells, gastric cancer tumor primary cell, micro- according to 1-1.5ul magnetic suspension
The ratio of grain cell every square centimeter is added, and the light culture bottle that shakes is uniformly distributed magnetic suspension particle in the medium.
Culture bottle is put back in incubator, so that cell is incubated for magnetic suspension particle and in conjunction with magnetic suspension particle, 37
Be incubated for 12h under the conditions of DEG C, discard the culture medium in culture bottle or culture dish, using pancreas enzyme -EDTA digestive juice (be purchased from Corning,
25-053-CI) vitellophag is added four into culture bottle or culture dish when cell falls off from culture dish or culture bottle bottom
The gastric cancer tumor primitive cell culture base of times pancreas enzyme -EDTA digestive juice volume, mixes and is transferred in centrifuge tube, 300rpm centrifugation
5 minutes, while magnetic driven device, 96 orifice plates and Cover Gasket with 70% alcohol spray disinfectant and are put into Biohazard Safety Equipment.
(contain 10%noggin, 50%Wnt-3a, 1nM gastrin, 10% with the primary organoid culture medium of gastric cancer tumor
The DMEM/F12 culture medium of ITS-A, 1%N2,2%B-27,100ng/ml EGF, 200ng/ml FGF) cell is resuspended, it mixes simultaneously
Cell in centrifuge tube is counted；
Feeder cells and gastric cancer tumor primary cell are inoculated with according to 2: 1 ratio, so that the every hole of 96 orifice plates is thin
Born of the same parents' number is 5000, liquid volume 75ul, shakes gently 96 orifice plates and the culture medium being added is uniformly distributed in hole.
Cover Gasket, magnetic driven device and 96 orifice plate lids are successively covered on 96 orifice plates, attached drawing 1 is shown in specific setting, by 96 holes
Culture plate is put into incubator and cultivates, and every other day replaces fresh growth medium, final to obtain the primary organoid of gastric cancer tumor.
Wherein the replacing options of culture medium specifically include: 96 orifice plates being removed from incubator, and are sprayed with 70% alcohol
Orifice plate outer surface moves into Biohazard Safety Equipment；It successively opens 96 orifice plate lids, magnetic driven device and Cover Gasket and overturns placement；It will
Magnetic driven device is placed in 96 orifice plate bottoms, and 3D cell coculture should be attracted by magnetic force and be moved to orifice plate bottom at this time, light to shake
96 orifice plates make 3D coculture offset bore center, and removing culture medium at this time prevents the damage of 3D coculture, and new training is added
Base is supported, the magnetic driven device of 96 orifice plate bottoms is removed, Cover Gasket, magnetic driven device and 96 orifice plate lids is successively covered on 96 orifice plates,
It is put into incubator and cultivates.
Performance characterization and analysis:
The feeder cells of acquisition, gastric cancer tumor primary cell and the primary organoid of gastric cancer tumor are carried out using microscope
Observation, the morphosis of each cell such as Fig. 2-4, by above-mentioned three figure it is found that the gastric cancer tumor obtained by cultivation of the invention
The structure of primary organoid and feeder cells, gastric cancer tumor cell is entirely different, wherein feeder cells, gastric cancer tumor cell
It can be particularly seen the state of each cell, i.e., the cell that the two culture obtains is cell monolayer, and the primary class device of gastric cancer tumor
Official can not observe the form of its cell, be the organoid with certain stereochemical structure, not cell monolayer.
Identification and analysis is carried out to the primary organoid of gastric cancer tumor, the specific method is as follows:
The primary organoid of the gastric cancer tumor of above-described embodiment culture is drawn in 15ml centrifuge tube from 96 orifice plates, 300g from
It the heart 3 minutes, is carefully inhaled with 1ml pipette and abandons supernatant；
The collagenase type I that 2ml 0.1% is added into 15ml centrifuge tube is digested, and 37 C water baths are incubated for 4 minutes,
Cell is gently blown and beaten using pipette, 300g is centrifuged 3 minutes, discards supernatant；
5ml Hank ' s balanced salt solution is added into 15ml centrifuge tube, gently blows and beats cell using pipette, 300g from
It the heart 3 minutes, discards supernatant.
Cell mass is resuspended and is counted using 3ml Hank ' S balanced salt solution, experimental group is according to 1*105It is a thin
Born of the same parents/400ul Hank ' S balanced salt solution/mouse inoculum concentration to C57BL/6 carry out tail vein injection, control group according to
400ul Hank ' S balanced salt solution/mouse carries out tail vein injection to C57BL/6.Mouse stomach is solved after 20 days
It cuts open, using enzyme-linked immunization to CA72-4 (cancer antigen), CEA (carcinomebryonic antigen), CA19-9 (sugar antigen) identification.It tests knot
Fruit is as shown in table 1.
Table 1
| Group | Experimental group | Control group |
| CA72-4(U/ml) | 32 | 0.5 |
| CEA(U/ml) | 120 | 12 |
| CA19-9(ng/ml) | 17.5 | 3.0 |
According to above-mentioned experimental result it is found that the primary organoid of gastric cancer tumor obtained using the present embodiment is by handling and matching
It is set to injection and the injection without above-mentioned cell is inoculated with mouse, in terms of the antigenic content of its stomach, experimental group
Each antigenic content obtained is much larger than the antigenic content of comparative example, is that gastric cancer is swollen using technical solution of the present invention acquisition therefore
The primary organoid of tumor.
Embodiment described above is merely preferred embodiments of the present invention, but protection scope of the present invention not office
Be limited to this, anyone skilled in the art within the technical scope of the present invention, according to the technique and scheme of the present invention
And its design is subject to equivalent substitution or change, should be covered by the scope of protection of the present invention.
Claims (10)
1. a kind of primary organoid cultural method of gastric cancer tumor, which is characterized in that described method includes following steps:
Feeder cells, gastric cancer tumor primary cell are obtained using enzyme digestion-cell culture method respectively；
The feeder cells, the gastric cancer tumor primary cell that culture density is 75%-85% are chosen, it is micro- to be separately added into magnetic suspension
Grain is uniformly mixed and is placed in incubation 10-12h in incubator；
Feeder cells, gastric cancer tumor primary cell after taking the incubation, are separately added into pancreas enzyme -EDTA-digestive juice and disappear
Cell culture medium is added after change to mix, and it is primary thin to be centrifuged feeder cells, gastric cancer tumor of the 4-8min acquisition containing magnetic particle
Born of the same parents；
The feeder cells containing magnetic particle, the gastric cancer tumor primary cell containing magnetic particle are seeded to gastric cancer tumor original
It for organoid culture medium, is cultivated 15-25 days under the conditions of magnetic drive, obtains the primary organoid of gastric cancer tumor.
2. the primary organoid cultural method of gastric cancer tumor according to claim 1, which is characterized in that described to use enzymic digestion
The step of method-cell culture method acquisition feeder cells, specifically includes:
The Tumor-surrounding tissue for obtaining solid tumor, shreds after using Tumor-surrounding tissue described in Hank ' s balanced salt solution rinse；
II Collagenase Type is added in Tumor-surrounding tissue after described shred and is incubated for 35-60min, obtains feeder cells suspension；
The feeder cells suspension is centrifuged, and cell is resuspended using feeder cells culture medium, inoculated and cultured obtains
Obtain feeder cells；
The feeder cells culture medium is the DMEM culture medium containing 10% people's AB serum.
3. the primary organoid cultural method of gastric cancer tumor according to claim 1, which is characterized in that described to use enzymic digestion
The step of method-cell culture method acquisition gastric cancer tumor primary cell, specifically includes:
The gastric cancer tumor tissue for obtaining stomach solid tumor, cuts after using gastric cancer tumor tissue described in Hank ' s balanced salt solution rinse
It is broken；
IV Collagenase Type is added in gastric cancer tumor tissue after described shred and is incubated for 100-150min, it is primary to obtain gastric cancer tumor
Cell suspending liquid；
The gastric cancer tumor primary cell suspension is centrifuged, and outstanding thin using gastric cancer tumor primitive cell culture base weight
Born of the same parents, inoculated and cultured obtain gastric cancer tumor primary cell；
The gastric cancer tumor primitive cell culture base is to contain number of people albumen, Wnt-3a albumen, gastrin, ITS-A, epidermal growth
The DMEM/F12 culture medium of the factor, fibroblast growth factor.
4. according to right want 1 described in the primary organoid cultural method of gastric cancer tumor, which is characterized in that it is described will be described containing magnetism
The feeder cells of particle, the gastric cancer tumor primary cell containing magnetic particle are seeded to the primary organoid culture medium of gastric cancer tumor
On, it cultivates 15-25 days, is obtained in gastric cancer tumor primary organoid step under the conditions of magnetic drive, it is described containing magnetic particle
Feeder cells and the ratio of the gastric cancer tumor primary cell quantity containing magnetic particle are 1.5-2.5: 1.
5. according to right want 1 described in the primary organoid cultural method of gastric cancer tumor, which is characterized in that it is described will be described containing magnetism
The feeder cells of particle, the gastric cancer tumor primary cell containing magnetic particle are seeded to the primary organoid culture medium of gastric cancer tumor
In, the cell number of each culture medium inoculated is 4000-6000.
6. according to right want 1 described in the primary organoid cultural method of gastric cancer tumor, which is characterized in that the gastric cancer tumor is primary
Organoid culture medium is raw containing number of people albumen, Wnt-3a albumen, gastrin, ITS-A, epidermal growth factor, fibroblast
The DMEM/F12 culture medium of the long factor.
7. the primary organoid cultural method of gastric cancer tumor according to claim 1, which is characterized in that the selection culture is close
Degree is the feeder cells, the gastric cancer tumor primary cell of 75%-85%, is separately added into magnetic suspension particle, is uniformly mixed simultaneously
It is placed in incubator and is incubated in 10-12h step, the magnetic suspension particle is according to 1-1.5ul/cm2The ratio of cell is added.
8. the primary organoid cultural method of gastric cancer tumor according to claim 1, which is characterized in that the incubation process
Temperature is 36-38 DEG C.
9. the primary organoid cultural method of gastric cancer tumor according to claim 2, which is characterized in that the feeder cells
Inoculum density be 0.8*105-1.2*105A/ml.
10. the primary organoid cultural method of gastric cancer tumor according to claim 3, which is characterized in that the gastric cancer tumor
The inoculum density of primary cell is 5*105-8*105A/ml.
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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 |
| WO2023060710A1 (en) * | 2021-10-15 | 2023-04-20 | 合肥中科普瑞昇生物医药科技有限公司 | Culture medium and culture method for gastric cancer organoids |
| 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 |
| CN118028229A (en) * | 2024-04-11 | 2024-05-14 | 成都云测医学生物技术有限公司 | Preparation method for obtaining mesenchymal stem cells from nerve organoids |
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