CN109207428A - The separation of circulating tumor cell and cultural method - Google Patents
The separation of circulating tumor cell and cultural method Download PDFInfo
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Abstract
The invention proposes the separation of circulating tumor cell and cultural method, screens the method for drug, determines the method and culture medium of drug effectiveness.The separation of the circulating tumor cell and cultural method include: the CD45 positive cell removed in anticoagulation cirumferential blood using Human CD45 Depletion Cocktail kit, to obtain CD45 negative cells liquid；The CD45 negative cells liquid is handled using density-gradient centrifugation method, the cell liquid on upper layer and middle layer after collecting centrifugation；And the cell in the cell liquid is cultivated.Using the separation of circulating tumor cell of the present invention and cultural method can effectively enrichment cycles tumour cell, yield is higher, and purity is preferable, easy to operate, is suitable for scale application.
Description
Technical field
The present invention relates to biological fields.In particular it relates to separation and the cultural method of circulating tumor cell.
Background technique
Metastases are the main reason for leading to oncotherapy failure and dead tumor patient.Malignant cell occurs to turn
The mode of shifting mainly has 4 kinds: 1. directly invading surrounding tissue, 2. Lymph Node Metastasis, 3. hematogenous metastasis, 4. fall off Implantation matastasis.
Wherein hematogenous metastasis refers to that tumour cell is exuded in the tissue around primary tumo(u)r, and enters blood vessel, and it is thin to form circulating tumor
Born of the same parents (circulating tumor cells, CTC), and it is transferred to lung, liver, Nao Deng remote organization with blood flow, then ooze out and adapt to
New microenvironment forms transfer stove.Therefore, the CTC in early detection blood, for patient's Index for diagnosis, therapeutic evaluation and a
Bodyization treatment suffers from important directive function.
Experiment shows the CTCs type of tumor patient, the situation of change of number, be study tumor disease process it is important according to
According to the appearance of CTCs is also closely related with the prognosis of patient with advanced cancer, and it is current for carrying out prognosis evaluation to metastatic tumour patient
The widest field of CTC clinical application.Meanwhile CTCs detection can obtain at any time as a kind of simple and small wound blood testing
Take and be used to assess the prognosis of patient.
However, still requiring study for the separation of circulating tumor cell and cultural method at present.
Summary of the invention
The present invention is directed to solve at least one the technical problems existing in the prior art at least to a certain extent.
For this purpose, in one aspect of the invention, the invention proposes a kind of separation of circulating tumor cell and cultural methods.
According to an embodiment of the invention, the described method includes: being removed using Human CD45Depletion Cocktail kit anti-
CD45 positive cell in solidifying peripheral blood, to obtain CD45 negative cells liquid；Using density-gradient centrifugation method to the CD45
Negative cells liquid is handled, the cell liquid on upper layer and middle layer after collecting centrifugation；And to the cell in the cell liquid
It is cultivated.
Since CTCs is usually in CD45-, therefore the CD45 in peripheral blood is removed using kit+Cell, to avoid non-CTCs
It causes to extract and interfere.But inventors have found that if CD45 is removed only with kit+Cell still cannot effectively be enriched with CTCs,
Heteroproteose cell is on the high side.Then, inventor carries out density gradient centrifugation to the CD45 negative cells liquid handled through kit, utilizes
The different characteristic of different cell mass specific gravity, isolates CTCs in peripheral blood.After being centrifuged, liquid be divided into upper layer, middle layer, under
Layer, CTCs are present in middle layer mostly.However, it is found by the inventors that being also easy to be mixed with a small amount of CTCs in upper liquid.Therefore, it sends out
Bright people has collected the cell liquid of upper layer and middle layer, and cultivates it, filter out be capable of normal growth circulating tumor it is thin
Born of the same parents.The separation of circulating tumor cell according to an embodiment of the present invention and cultural method being capable of effectively enrichment cycles tumours as a result,
Cell, yield is higher, and purity is preferable, easy to operate, is suitable for scale application.
According to an embodiment of the invention, culture medium used by the culture includes: Wnt 3a albumen, N- mucolyticum
Acid, gastrins, epithelical cell growth factor, R-spondin1 albumen, noggin, Fibroblast growth factor-10, TGF-β
RI, ALK4 and/or ALK7 inhibitor, antibacterial agent, niacinamide, DMEM culture medium and F12 culture medium.
According to an embodiment of the invention, final concentration of 80~200ng/mL of the Wnt 3a albumen, N- mucolyticum
Final concentration of 0.5~the 2mM, final concentration of 0.5~2nM of gastrins, final concentration of the 30 of epithelical cell growth factor of acid
Final concentration of 300~800ng/mL of~70ng/mL, R-spondin1 albumen, final concentration of 80~120ng/mL of noggin,
Final concentration of 5~15ng/mL of Fibroblast growth factor-10, the final concentration of TGF-β RI, ALK4 and/or ALK7 inhibitor
It is 1~5 μM, final concentration of 50~150 μ g/mL of antibacterial agent, final concentration of 5~20mM of niacinamide, DMEM culture medium and F12
The volume ratio of culture medium is 1:1.
According to an embodiment of the invention, the culture medium further comprises: Rock inhibitor.
According to an embodiment of the invention, final concentration of 5~10 μm of ol/L of the Rock inhibitor.
According to an embodiment of the invention, the culture includes: the cell and liquid Matrigel base taken in the cell liquid
The mixing of matter glue, and obtained mixed liquor is added in orifice plate, preheating, to solidify the matrigel；To the solidification
The culture medium is added in matrigel, cultivates 10~12 days；Or cell and the culture medium in the cell liquid is taken to carry out
Mixing, and obtained culture solution is added dropwise to bottom and is covered in the orifice plate of rat-tail source collagen I, it cultivates 10~12 days.
According to an embodiment of the invention, the density-gradient centrifugation method is carried out using following manner: by the CD45
Negative cells liquid is mixed with the PBS buffer solution containing 1~5%FBS, obtains mixed liquor；By the mixed liquor with (1~5):
1 ratio is layered on lymphocyte separation medium, is then centrifuged 8~15min with the revolving speed of 1000~1500g, lifting speed is respectively 1
With 0.
According to an embodiment of the invention, the peripheral blood is derived from the biology with cancer, the cancer includes gastric cancer, intestines
Cancer, lung cancer, gallbladder cancer or cancer of pancreas.
In still another aspect of the invention, the invention proposes a kind of methods for screening drug.According to an embodiment of the invention,
The described method includes: drug candidate is contacted with circulating tumor cell；Measure the biology of contact front and back circulating tumor cell
Activity, compared with before contact, it is the drug candidate as target that the bioactivity of the circulating tumor cell, which reduces, after contact
The instruction of drug, wherein the circulating tumor cell is the separation and cultural method institute using circulating tumor cell noted earlier
What separation and culture obtained.The medicine that can accurately filter out inhibition CTCs according to the method for the embodiment of the present invention is utilized as a result,
Object, to play the effect for the treatment of cancer.
According to an embodiment of the invention, the drug is used for treating cancer.
In still another aspect of the invention, the invention proposes a kind of methods of determining drug effectiveness.It is according to the present invention
Embodiment, which comprises contact drug with circulating tumor cell, the drug is suitable for making the life of circulating tumor cell
Object activity reduces；The bioactivity for measuring contact front and back circulating tumor cell, compared with before contact, the circulation after contact
The bioactivity of tumour cell reduces, and is that the drug effectively indicates, wherein the circulating tumor cell is to utilize front institute
It states the separation of circulating tumor cell and cultural method is separated and culture obtains.As a result, using according to an embodiment of the present invention
Method can accurately determine whether drug has the effect of inhibiting CTCs, to play the effect for the treatment of cancer.
According to an embodiment of the invention, the drug is used for treating cancer.
In still another aspect of the invention, the invention proposes a kind of culture mediums.According to an embodiment of the invention, the culture
Base includes: the Wnt 3a albumen of 80~200ng/mL；The N-acetylcystein of 0.5~2mM；The gastrins of 0.5~2nM；
The epithelical cell growth factor of 30~70ng/mL；The R-spondin1 albumen of 300~800ng/mL；The head of 80~120ng/mL
Albumen；The Fibroblast growth factor-10 of 5~15ng/mL；1~5 μM of TGF-β RI, ALK4 and/or ALK7 inhibitor；50
The antibacterial agent of~150 μ g/mL；The niacinamide of 5~20mM；And DMEM culture medium and F12 culture medium, volume ratio 1:1.By
This, can make circulating tumor cell normal growth using culture medium according to an embodiment of the present invention.
According to an embodiment of the invention, the culture medium further comprises: Rock inhibitor.
According to an embodiment of the invention, the concentration of the Rock inhibitor is 5~10 μm of ol/L.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Detailed description of the invention
Above-mentioned and/or additional aspect of the invention and advantage will become from the description of the embodiment in conjunction with the following figures
Obviously and it is readily appreciated that, in which:
Fig. 1 shows the separation of circulating tumor cell according to an embodiment of the invention and the process signal of cultural method
Figure；
Fig. 2 shows the flow diagram of the method for screening drug according to an embodiment of the invention；
Fig. 3 shows the flow diagram of the method for determining drug effectiveness according to an embodiment of the invention；
Fig. 4 shows 4 × microscope figure of matrigel culture circulating tumor cell according to an embodiment of the invention；
Fig. 5 shows the 4 of the matrigel culture circulating tumor cell of positive controls according to an embodiment of the invention
× microscope figure；
Fig. 6 shows the 4 of the matrigel culture circulating tumor cell of negative control group according to an embodiment of the invention
× microscope figure；
Fig. 7 show collagen according to an embodiment of the invention coating bottom plate culture circulating tumor cell 4 × it is micro-
Mirror figure；
Fig. 8 shows point for the influence that ROCK inhibitor according to an embodiment of the invention grows circulating tumor cell
Analyse schematic diagram；
Fig. 9 shows medicament sensitivity analysis schematic diagram according to an embodiment of the invention.
Specific embodiment
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this hair
It is bright, and be not considered as limiting the invention.
The invention proposes the separation of circulating tumor cell and cultural method, the method for screening drug, determine that drug is effective
The method and culture medium of property, will be described in greater detail respectively below.
The separation of circulating tumor cell and cultural method
In one aspect of the invention, the invention proposes a kind of separation of circulating tumor cell and cultural methods.According to
The embodiment of the present invention, referring to Fig. 1, this method comprises:
S100 kit removes CD45 positive cell
In this step, it is removed in anticoagulation cirumferential blood using Human CD45Depletion Cocktail kit
CD45 positive cell, to obtain CD45 negative cells liquid.Since CTCs is usually in CD45-, therefore it is outer using kit removing
CD45 in all blood+Cell is caused to extract and be interfered to avoid non-CTCs.
According to an embodiment of the invention, peripheral blood is derived from the biology with cancer, cancer includes gastric cancer, intestinal cancer, lung cancer, gallbladder
Capsule cancer or cancer of pancreas.The circulating tumor cell that above-mentioned cancer can be obtained using method of the invention as a result, to Index for diagnosis,
Therapeutic evaluation and individualized treatment play a significant role.
It should be noted that the present invention does not do considered critical for the type of biology, people, pig, dog, mouse, rabbit, sheep can be
Deng specifically can flexible choice according to the actual situation.
S200 density gradient centrifugation
In this step, CD45 negative cells liquid is handled using density-gradient centrifugation method, it is upper after collecting centrifugation
The cell liquid of layer and middle layer.Inventors have found that if removing CD45 only with kit+Cell cannot effectively be enriched with CTCs, miscellaneous
Cell is on the high side.Then, inventor carries out density gradient centrifugation to the CD45 negative cells liquid that handles through kit, using outer
The different characteristic of different cell mass specific gravity, isolates CTCs in all blood.After being centrifuged, liquid is divided into upper layer, middle layer, lower layer,
CTCs is present in middle layer mostly.However, it is found by the inventors that being also easy to be mixed with a small amount of CTCs in upper liquid.Therefore, inventor
The cell liquid of upper layer and middle layer is had collected, to improve the yield of CTCs.
According to an embodiment of the invention, density-gradient centrifugation method is carried out using following manner: by CD45 negative cells
Liquid is mixed with the PBS buffer solution containing 1~5%FBS, obtains mixed liquor；By mixed liquor with (1~5): 1 ratio is layered on
On lymphocyte separation medium, 8~15min is then centrifuged with the revolving speed of 1000~1500g, lifting speed is respectively 1 and 0.As a result, with
Just CTCs is further separated.
S300 culture
In this step, the cell in cell liquid is cultivated.
It is cultivated by the cell in the cell liquid on upper layer and middle layer to collection, filters out and be capable of normal growth
Circulating tumor cell.The separation of circulating tumor cell according to an embodiment of the present invention and cultural method can be effectively rich as a result,
Collecting circulating tumor cell, yield is higher, and purity is preferable, and it is easy to operate, it is suitable for scale application.
According to an embodiment of the invention, culture medium used by cultivating includes: Wnt 3a albumen, N-acetylcystein
(N-acetylcysteine), gastrins (Gastrin), epithelical cell growth factor (EGF), R-spondin1 albumen, head
Albumen (Noggin), Fibroblast growth factor-10 (FGF10), TGF-β RI, ALK4 and/or ALK7 inhibitor (A83-01),
Antibacterial agent (Normocin), niacinamide (Nicotinamide), DMEM culture medium and F12 culture medium.It is according to the present invention specific
Embodiment, final concentration of 80~200ng/mL of Wnt 3a albumen, final concentration of 0.5~2mM of N-acetylcystein promote stomach
Final concentration of 0.5~2nM of liquid element, final concentration of 30~70ng/mL of epithelical cell growth factor, R-spondin1 albumen
Final concentration of 300~800ng/mL, final concentration of 80~120ng/mL of noggin, the end of Fibroblast growth factor-10 are dense
Spending is 5~15ng/mL, final concentration of 1~5 μM of TGF-β RI, ALK4 and/or ALK7 inhibitor, final concentration of the 50 of antibacterial agent
The volume ratio of~150 μ g/mL, final concentration of 5~20mM of niacinamide, DMEM culture medium and F12 culture medium is 1:1.
It should be noted that the concentration of each factor is based on DMEM culture medium/F12 training in culture medium described in the invention
For the total volume for supporting base.
Inventor, which obtains above-mentioned culture medium composition and proportion, CTCs by many experiments, normally to be given birth in the culture medium
It is long, and other cells growth conditions in the culture medium are bad, the culture medium not only acts as the purpose of screening, removal of impurities as a result, with
Just CTCs is purified, also can reach the purpose of enrichment CTCs.
According to an embodiment of the invention, culture medium further comprises: Rock inhibitor.
Rho-Rock is a special signal path, it forms and interaction etc. between stem cell cell colony
It plays an important role.When the signal path is by ROCK inhibitor, after blocking such as Y-27632, the normal Colony forming of stem cell can quilt
Weaken significantly.Inventors be surprised to learn that Rock inhibitor can effectively promote the growth of circulating tumor cell, reach preferable
It is enriched with purpose.However, CTCs growth conditions are bad in the culture medium without Rock inhibitor.To which Rock inhibitor adds
Adding can further achieve the purpose that optimize circulating tumor cell cultivating system.Wherein, final concentration of the 5~10 of Rock inhibitor
When μm ol/L, effect is preferable, and obtained circulating tumor cell quantity is more, and purity is higher.
It should be noted that the present invention does not do considered critical for the training method of CTCs, it can be clever according to actual needs
Selection living, preferably with the training method of 3D culture.For example, mode 1: taking the cell and liquid Matrigel matrix in cell liquid
Glue mixing, and obtained mixed liquor is added in orifice plate, preheating, to solidify matrigel；Add into the matrigel of solidification
Enter culture medium, cultivates 10~12 days；Mode 2: taking the cell in cell liquid to be mixed with culture medium, and by obtained culture
Drop adds to bottom and is covered in the orifice plate of rat-tail source collagen I, cultivates 10~12 days.Inventors have found that being coated with compared to collagen
The training method (mode 2) of bottom plate, the effect using Matrigel matrigel culture (mode 1) are more preferable.
The method for screening drug
In another aspect of this invention, the invention proposes a kind of methods for screening drug.According to an embodiment of the invention,
Referring to fig. 2, this method comprises:
A100 contacts drug candidate with circulating tumor cell
In this step, drug candidate is contacted with circulating tumor cell.
The bioactivity of A200 measurement contact front and back circulating tumor cell
In this step, the bioactivity of measurement contact front and back circulating tumor cell recycles after contact compared with before contact
The bioactivity of tumour cell reduces, and is instruction of the drug candidate as drug target.
According to an embodiment of the invention, circulating tumor cell is separation and the cultural method using previous cycles tumour cell
What separated and culture obtained.
The exploitation of tumor-targeting drug at present there is no mostly using entity tumor as the target for the treatment of using CTCs as targeting
Drug.It can be used for screening having using the separation of circulating tumor cell of the invention and cultural method CTCs obtained and resist
The drug of tumor effect.After contacting with anti-tumor drug with CTCs, the growth metabolism of CTCs can be inhibited, to pass through detection
The bioactivity of CTCs can reach the purpose of antitumor medicine screening.
It should be noted that the present invention does not do considered critical for the Testing index of " bioactivity ", as long as being related to thin
Intracellular growth metabolic capability can be detected.For example, the half lethal dose etc. of the expression quantity of gene or albumen, cell.
In addition, term " screening drug " used in the present invention should broadly understood, either the type of screening drug,
The drug being applicable in for specified disease；Be also possible to screen drug dosage, due to different sufferers may be directed to it is same
The sensibility of drug is different, and the drug dose for causing it to be applicable in is different.
According to an embodiment of the invention, the drug is used for treating cancer.Utilize the separation of circulating tumor cell of the invention
It can be used for screening the drug with antitumous effect with cultural method CTCs obtained.
It will be appreciated to those of skill in the art that described by separation and cultural method above for circulating tumor cell
Feature and advantage, the method for being equally applicable to the screening drug, details are not described herein.
The method for determining drug effectiveness
In still another aspect of the invention, the invention proposes a kind of methods of determining drug effectiveness.It is according to the present invention
Embodiment, referring to Fig. 3, this method comprises:
P100 contacts drug with circulating tumor cell
In this step, drug is contacted with circulating tumor cell, drug is suitable for making the biology of circulating tumor cell living
Property reduce.
The bioactivity of P200 measurement contact front and back circulating tumor cell
In this step, the bioactivity of measurement contact front and back circulating tumor cell recycles after contact compared with before contact
The bioactivity of tumour cell reduces, and is that drug effectively indicates, wherein circulating tumor cell is swollen using circulation noted earlier
The separation of oncocyte and cultural method is separated and culture obtains.
The drug for having validity can be such that the bioactivity of circulating tumor cell reduces, thus, make the drug and circulation
Tumour cell contact may indicate that the drug is effective if the bioactivity of circulating tumor cell reduces after contact.
According to an embodiment of the invention, the drug is used for treating cancer.
It will be appreciated to those of skill in the art that described by separation and cultural method above for circulating tumor cell
Feature and advantage, the method for being equally applicable to the determination drug effectiveness, details are not described herein.
Culture medium
In still another aspect of the invention, the invention proposes a kind of culture mediums.According to an embodiment of the invention, the culture medium
It include: the Wnt 3a albumen of 80~200ng/mL；The N-acetylcystein of 0.5~2mM；The gastrins of 0.5~2nM；30
The epithelical cell growth factor of~70ng/mL；The R-spondin1 albumen of 300~800ng/mL；The head egg of 80~120ng/mL
It is white；The Fibroblast growth factor-10 of 5~15ng/mL；1~5 μM of TGF-β RI, ALK4 and/or ALK7 inhibitor；50~
The antibacterial agent of 150 μ g/mL；The niacinamide of 5~20mM；And DMEM culture medium and F12 culture medium, volume ratio 1:1.CTCs energy
Enough normal growths in the culture medium, and other cells growth conditions in the culture medium are bad, the culture medium not only rises as a result,
To the purpose of screening, removal of impurities, to purify CTCs, the purpose of enrichment CTCs also can reach.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or item are not specified in embodiment
Part, it described technology or conditions or is carried out according to the literature in the art according to product description.Agents useful for same or instrument
Production firm person is not specified in device, and being can be with conventional products that are commercially available.
The culture (3D culture) of 1 tumour patient Peripheral Circulation tumour cell of embodiment
1, the enrichment of peripheral blood tumour cell
1) PBS containing 2%FBS is prepared；
2) it is derived from the Human CD45Depletion that 50 μ l/ml are added in the fresh anticoagulation of tumour patient
Cocktail, (15~25 DEG C) incubation 20min of room temperature；
3) it is added and the isometric lymphocyte separation medium of blood sample (Ficoll) in new centrifuge tube；
4) after being incubated at room temperature, isometric PBS containing 2%FBS is added in blood sample, after mixing, by cell liquid with volume
It is layered on lymphocyte separation medium than 2:1；
5) 1200g, 10min, lifting speed are respectively 1 and 0, centrifugation；
6) after being centrifuged, the monocyte of the CD45 feminine gender of upper layer and middle layer is collected, 3~5 times of volumes are added contains 2%
The PBS of FBS is suspended.300g, 10min, lifting speed 1 and 0, centrifugation；
7) it cleans 2 times and counts according to step 6).
2, the culture of tumour patient Peripheral Circulation tumour cell
1) cell that enrichment obtains is divided into two parts, a copy of it is according to count density in the cell precipitation of enrichment
Suitable Matrigel matrigel is added, after mixing, is added dropwise in 24 orifice plates with 50 holes μ l/.CO210min is preheated in incubator,
Until matrigel solidifies；
2) addition culture medium, 37 DEG C, 5%CO2It is cultivated in incubator, the ingredient of culture medium is as shown in table 1；
1 culture medium of table composition
| Reagent name | Final concentration |
| Wnt 3a | 100ng/ml |
| N-acetylcysteine | 1mM |
| Gastrin | 1nM |
| EGF | 50ng/ml |
| Rspondin1 | 500ng/ml |
| Noggin | 100ng/ml |
| FGF10 | 10ng/ml |
| A83-01 | 2μM |
| Normocin | 100μg/ml |
| Nicotinamide | 10mM |
| DMEM/F12 | 1:1 |
3) every 3 days replacement cell culture mediums.At culture 10-12 days, the growth conditions of cell are as shown in Figure 4.This patient
Circulating tumor cell is extracted in blood, is placed in matrigel after cultivating, single tumor cell long balling-up in 3D matrix structure
Shape tumor tissues analog (PDTA) co-cultures in this patient and obtains 7 PDTA.
The culture (3D culture) of 2 tumour patient Peripheral Circulation tumour cell of embodiment
In this embodiment, it is enriched with according to the method for embodiment 1 and cultivates circulating tumor cell, difference is, culture medium
As shown in table 2.
At culture 10-12 days, the formation of tumour analog can be seen, the growth conditions of cell are good, and tumour is similar
The formation efficiency and growing state of object, it is similar with the cultivating system in embodiment 1, obtain 6 PDTA.
2 culture medium of table composition
| Reagent name | Final concentration |
| Wnt 3a | 200ng/ml |
| N-acetylcysteine | 1.2mM |
| Gastrin | 2nM |
| EGF | 60ng/ml |
| Rspondin1 | 800ng/ml |
| Noggin | 120ng/ml |
| FGF10 | 15ng/ml |
| A83-01 | 3μM |
| Normocin | 150μg/ml |
| Nicotinamide | 15mM |
| DMEM/F12 | 1:1 |
The extraction of 3 Peripheral Circulation tumour cell of embodiment and cultural method establish (3D culture, positive control)
1, the enrichment of peripheral blood tumour cell
1) PBS containing 2%FBS is prepared；
2) 1000 gastric cancer tumor cells of addition in the fresh anticoagulation of normal person are derived from, are added in anticoagulation again later
The Human CD45Depletion Cocktail of 50 μ l/ml, (15~25 DEG C) incubation 20min of room temperature；
3) it is added and the isometric lymphocyte separation medium of blood sample (Ficoll) in new centrifuge tube；
4) after being incubated at room temperature, isometric PBS containing 2%FBS is added in blood sample, after mixing, by cell liquid with volume
It is layered on lymphocyte separation medium than 2:1；
5) 1200g, 10min, lifting speed are respectively 1 and 0, centrifugation；
6) after being centrifuged, the monocyte of the CD45 feminine gender of upper layer and middle layer is collected, 3~5 times of volumes are added contains 2%
The PBS of FBS is suspended.300g, 10min, lifting speed 1 and 0, centrifugation；
7) it cleans 2 times and counts according to step 6).
2, the culture (3D culture) of Peripheral Circulation tumour cell
1) suitable Matrigel matrigel is added in the cell precipitation of enrichment according to count density, after mixing, with 50 μ
The hole l/ is added dropwise in 24 orifice plates.CO210min is preheated in incubator, until matrigel solidifies；
2) addition culture medium, 37 DEG C, 5%CO2It is cultivated in incubator.Medium component is as shown in table 1；
3) every 3 days replacement cell culture mediums.At culture 10-12 days, the growth conditions of cell as shown in figure 5, PDTA shape
It is about 60% at efficiency.
The culture (3D culture, negative control) of 4 normal human peripheral blood's circulating tumor cell of embodiment
1, the enrichment of peripheral blood tumour cell
1) PBS containing 2%FBS is prepared；
2) it is derived from the Human CD45Depletion that 50 μ l/ml are added in the fresh anticoagulation of normal person
Cocktail, (15~25 DEG C) incubation 20min of room temperature；
3) it is added and the isometric lymphocyte separation medium of blood sample (Ficoll) in new centrifuge tube；
4) after being incubated at room temperature, isometric PBS containing 2%FBS is added in blood sample, after mixing, by cell liquid with volume
It is layered on lymphocyte separation medium than 2:1；
5) 1200g, 10min, lifting speed are respectively 1 and 0, centrifugation；
6) after being centrifuged, the monocyte of the CD45 feminine gender of upper layer and middle layer is collected, 3~5 times of volumes are added contains 2%
The PBS of FBS is suspended.300g, 10min, lifting speed 1 and 0, centrifugation；
7) it cleans 2 times and counts according to step 6).
2, the culture (3D culture) of normal human peripheral blood's circulating tumor cell
1) suitable Matrigel matrigel is added in the cell precipitation of enrichment according to count density, after mixing, with 50 μ
The hole l/ is added dropwise in 24 orifice plates.CO210min is preheated in incubator, until matrigel solidifies；
2) addition culture medium, 37 DEG C, 5%CO2It is cultivated in incubator.The ingredient of culture medium is as shown in table 1；
3) every 3 days replacement cell culture mediums.At culture 10-12 days, the growth conditions of cell are as shown in Figure 6.Normal person's blood
Without circulating tumor cell in clear, so the formation of tumor tissues analog (PDTA) is not seen in culture.
The culture (culture based on collagen coating bottom plate) of 5 tumour patient Peripheral Circulation tumour cell of embodiment
1, the enrichment of tumour patient Peripheral Circulation tumour cell
Enrichment and separation circulating tumor cell according to the method for embodiment 1.
2, collagen I is coated with culture plate, and suspend culture CTCs
1) collagen I that one layer of rat-tail source is spread in 24 orifice bore bottoms, is placed at room temperature for about 1 hour, is washed with DMEM culture medium
Go extra collagen I；
2) after the half cell count for obtaining step 1 enrichment, it is suspended to suitable concentration with culture medium, is directly added dropwise solidifying
Solid collagen I on, 37 DEG C, 5%CO2It is cultivated in incubator, every 3 days replacement cell culture mediums.At culture 10-12 days, as a result such as
Shown in Fig. 7, tumour cell is assembled, and forms the cell mass of irregular alignment, the tumour cell cultivated under the conditions of this cannot
Form the structure of 3D, culture effect deviation.
The influence that embodiment 6ROCK inhibitor grows circulating tumor cell
After the circulating tumor cell that enrichment obtains is carried out passage amplification, cultivated respectively with two kinds of culture mediums: (1) such as
Culture medium used in embodiment 1, obtained organoid are added 8 μm of ol/L's as shown in Fig. 8 A, 8B and 8C, in (2) culture medium
ROCK inhibitor (Y27632), the organoid cultivated such as Fig. 8 D, 8E, shown in 8F.In above-mentioned two situations, initial incubation is
The organoid species number that each cell culture well is added is essentially identical.After culture 1 day and 3 days, two kinds of condition of culture are counted respectively
Under organoid number as a result as shown in fig. 8g show that ROCK inhibitor Y27632 can be improved the survival rate of organoid, thus
Promote the growth of circulating tumor cell.
The detection of 7 peripheral blood CTCs drug susceptibility of embodiment
Below by taking the patients with gastric cancer CTCs of 1 mesostroma glue culture of embodiment as an example, illustrate that detection peripheral blood CTCs drug is quick
The method of perception.This method can be used for but be not limited to the drug susceptibility detection of peripheral blood from patients with gastric cancer CTCs, it can also be used to its
The detection of the peripheral blood CTCs drug susceptibility of his tumor types patient.
1, the passage of peripheral blood CTCs and bed board: disappear obtained peripheral blood CTCs cell is cultivated by embodiment 1
Change, is laid in 96 orifice plates again.
2, drug gradient experiment:
1) method of gradient dilution is used: after drawing 10 μ L, 5 μ L, 2.5 μ L, 2 μ L, the original content of 1 μ L or dilution respectively
Drug is added in the centrifuge tube of the patients with gastric cancer CTCs culture medium containing 1mL, then 0.5mL to the is drawn from above-mentioned centrifuge tube
Two have been loaded in the centrifuge tube of 0.5mL complete medium, i.e., dilute drug according to 1:1.Above method is repeated, is successively diluted,
Finally obtain 5 kinds of concentration needed for dosing；
2) control group draws the DMSO or DMF of 1 μ L, is added in the patients with gastric cancer CTCs culture medium of 1mL, a kind of concentration
Drug and experimental comparison group are each provided 2-3 multiple holes, in addition be arranged 3 multiple holes add CTCs culture medium, DMSO control and DMF pairs
According to it is 140 μ L that drug volume, which is added, in every hole；
3) change liquid and second of dosing: first time dosing changes liquid and second of dosing when being the 0th day, the 3rd day.Adding consistency
It is identical as first time；
4) the 5th day, the OD value of cell after dosing culture, OD value CCK-8 cytoactive detection: are detected with CCK8 detection reagent
Size reflect the effect of cell activity and drug to analog, every hole is added configured CCK8 and detects liquid, after mixing
After placing 37 DEG C of 3~4h of incubation, microplate reader detects light absorption value at 450nm and 650nm.Calculation formula is as follows: ODDelta=
ODBlank450-ODBlank650
Contributive rate (%)=(experimental group ODDelta- blank group ODDelta) × 100%/(control of the drug to organoid
Group ODDelta- blank group ODDelta)
As a result as shown in Figure 9: by taking 2 peripheral blood from patients with gastric cancer CTCs as an example, detecting it to chemotherapeutics oxaliplatin
Sensibility.Different patients are different to the drug susceptibility of oxaliplatin as the result is shown, oxaliplatin to YDY-S-002 patient half
Number lethal dose IC50 is 8.9 μM, and oxaliplatin is 31.9 μM to the half lethal dose IC50 of YDY-S-004 patient, i.e.,
YDY-S-002 patient is higher than YDY-S-004 patient to the sensibility of oxaliplatin.The peripheral blood of the different patients as the result is shown
CTCs is different to the sensibility of same drug, is enriched with using method of the invention and the CTCs of culture can be used for detecting patient to change
Treat the sensibility of drug.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field
Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples
It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, modifies, replacement and variant.
Claims (10)
1. separation and the cultural method of a kind of circulating tumor cell characterized by comprising
CD45 positive cell in anticoagulation cirumferential blood is removed using Human CD45 Depletion Cocktail kit, with
Just CD45 negative cells liquid is obtained；
The CD45 negative cells liquid is handled using density-gradient centrifugation method, the upper layer and middle layer after collecting centrifugation
Cell liquid；And
Cell in the cell liquid is cultivated.
2. the method according to claim 1, wherein culture medium used by the culture includes:
Wnt 3a albumen, N-acetylcystein, gastrins, epithelical cell growth factor, R-spondin1 albumen, head egg
White, Fibroblast growth factor-10, TGF-β RI, ALK4 and/or ALK7 inhibitor, antibacterial agent, niacinamide, DMEM culture medium
With F12 culture medium；
Optionally, final concentration of 80~200ng/mL of the Wnt 3a albumen, N-acetylcystein final concentration of 0.5~
2mM, final concentration of 0.5~2nM of gastrins, the final concentration of 30~70ng/mL, R- of epithelical cell growth factor
Final concentration of 300~800ng/mL of spondin1 albumen, final concentration of 80~120ng/mL of noggin, fibroblast are raw
Final concentration of 1~5 μM of final concentration of 5~15ng/mL of the long factor 10, TGF-β RI, ALK4 and/or ALK7 inhibitor, antibacterial
Final concentration of 50~150 μ g/mL of agent, final concentration of 5~20mM of niacinamide, the volume of DMEM culture medium and F12 culture medium
Than for 1:1；
Optionally, the culture medium further comprises: Rock inhibitor；
Optionally, final concentration of 5~10 μm of ol/L of the Rock inhibitor.
3. according to the method described in claim 2, it is characterized in that, the culture includes:
It takes the cell in the cell liquid to mix with liquid Matrigel matrigel, and orifice plate is added in obtained mixed liquor
In, preheating, to solidify the matrigel；The culture medium is added into the matrigel of the solidification, cultivates 10~12 days；
Or
It takes the cell in the cell liquid to be mixed with the culture medium, and obtained culture solution is added dropwise to bottom and is covered with
In the orifice plate of rat-tail source collagen I, cultivate 10~12 days.
4. the method according to claim 1, wherein the density-gradient centrifugation method is carried out using following manner
:
The CD45 negative cells liquid is mixed with the PBS buffer solution containing 1~5%FBS, obtains mixed liquor；
By the mixed liquor with (1~5): 1 ratio is layered on lymphocyte separation medium, then with the revolving speed of 1000~1500g
It is centrifuged 8~15min, lifting speed is respectively 1 and 0.
5. the method according to claim 1, wherein the peripheral blood is derived from the biology with cancer, the cancer
Disease includes gastric cancer, intestinal cancer, lung cancer, gallbladder cancer or cancer of pancreas.
6. a kind of method for screening drug characterized by comprising
Drug candidate is contacted with circulating tumor cell；
The bioactivity of contact front and back circulating tumor cell is measured,
Compared with before contact, it is the drug candidate as target that the bioactivity of the circulating tumor cell, which reduces, after contact
The instruction of drug,
Wherein, the circulating tumor cell is the separation and culture using any one of Claims 1 to 55 circulating tumor cell
Method is separated and culture obtains.
7. according to the method described in claim 6, it is characterized in that, the drug is used for treating cancer.
8. a kind of method of determining drug effectiveness characterized by comprising
Drug is contacted with circulating tumor cell, the drug is suitable for reducing the bioactivity of circulating tumor cell；
The bioactivity of contact front and back circulating tumor cell is measured,
Compared with before contact, it is that the drug effectively indicates that the bioactivity of the circulating tumor cell, which reduces, after contact,
Wherein, the circulating tumor cell is the separation and culture using any one of Claims 1 to 55 circulating tumor cell
Method is separated and culture obtains.
9. according to the method described in claim 8, it is characterized in that, the drug is used for treating cancer.
10. a kind of culture medium characterized by comprising
The Wnt 3a albumen of 80~200ng/mL；
The N-acetylcystein of 0.5~2mM；
The gastrins of 0.5~2nM；
The epithelical cell growth factor of 30~70ng/mL；
The R-spondin1 albumen of 300~800ng/mL；
The noggin of 80~120ng/mL；
The Fibroblast growth factor-10 of 5~15ng/mL；
1~5 μM of TGF-β RI, ALK4 and/or ALK7 inhibitor；
The antibacterial agent of 50~150 μ g/mL；
The niacinamide of 5~20mM；And
DMEM culture medium and F12 culture medium, volume ratio 1:1；
Optionally, the culture medium further comprises: Rock inhibitor；
Optionally, the concentration of the Rock inhibitor is 5~10 μm of ol/L.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628721A (en) * | 2019-05-30 | 2019-12-31 | 佛山市璞狄医疗科技有限公司 | Isolated culture method and kit for circulating tumor cells |
| CN111197031A (en) * | 2020-02-21 | 2020-05-26 | 复旦大学附属肿瘤医院 | Intestinal cancer organoid culture and passage method originated from circulating tumor cells |
| CN111411082A (en) * | 2020-03-26 | 2020-07-14 | 中山大学孙逸仙纪念医院 | Culture medium for culturing CD90posi cells and culture method thereof |
| CN111855541A (en) * | 2019-04-26 | 2020-10-30 | 朱德新 | Circulating tumor cell detection reagent, kit and detection method |
| CN112501121A (en) * | 2020-11-24 | 2021-03-16 | 益善生物技术股份有限公司 | Basic culture medium and culture method for circulating tumor cells |
| CN112760286A (en) * | 2019-11-04 | 2021-05-07 | 北京基石生命科技有限公司 | Method for culturing primary cells of brain tumor solid tumor |
| CN115125212A (en) * | 2022-06-17 | 2022-09-30 | 成都诺医德医学检验实验室有限公司 | Peripheral blood circulation tumor cell organoid culture method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008008515A2 (en) * | 2006-07-14 | 2008-01-17 | Aviva Biosciences Corporation | Methods and compositions for detecting rare cells from a biological sample |
| CN101684457A (en) * | 2009-07-27 | 2010-03-31 | 中国科学院广州生物医药与健康研究院 | Application of I type transforming growth factor receptor inhibitor in producing induced multi-potent stem cells and method thereof |
| CN102439135A (en) * | 2009-02-03 | 2012-05-02 | 荷兰皇家科学院 | Culture medium for epithelial stem cells and organoids comprising said stem cells |
| CN103866016A (en) * | 2014-03-07 | 2014-06-18 | 复旦大学附属中山医院 | Circulating tumor cell detection kit and application thereof |
| WO2017188890A1 (en) * | 2016-04-29 | 2017-11-02 | National University Of Singapore | Cell culture |
-
2018
- 2018-09-12 CN CN201811061720.3A patent/CN109207428B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008008515A2 (en) * | 2006-07-14 | 2008-01-17 | Aviva Biosciences Corporation | Methods and compositions for detecting rare cells from a biological sample |
| CN102439135A (en) * | 2009-02-03 | 2012-05-02 | 荷兰皇家科学院 | Culture medium for epithelial stem cells and organoids comprising said stem cells |
| CN101684457A (en) * | 2009-07-27 | 2010-03-31 | 中国科学院广州生物医药与健康研究院 | Application of I type transforming growth factor receptor inhibitor in producing induced multi-potent stem cells and method thereof |
| CN103866016A (en) * | 2014-03-07 | 2014-06-18 | 复旦大学附属中山医院 | Circulating tumor cell detection kit and application thereof |
| WO2017188890A1 (en) * | 2016-04-29 | 2017-11-02 | National University Of Singapore | Cell culture |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111855541A (en) * | 2019-04-26 | 2020-10-30 | 朱德新 | Circulating tumor cell detection reagent, kit and detection method |
| CN110628721A (en) * | 2019-05-30 | 2019-12-31 | 佛山市璞狄医疗科技有限公司 | Isolated culture method and kit for circulating tumor cells |
| CN112760286A (en) * | 2019-11-04 | 2021-05-07 | 北京基石生命科技有限公司 | Method for culturing primary cells of brain tumor solid tumor |
| CN111197031A (en) * | 2020-02-21 | 2020-05-26 | 复旦大学附属肿瘤医院 | Intestinal cancer organoid culture and passage method originated from circulating tumor cells |
| CN111411082A (en) * | 2020-03-26 | 2020-07-14 | 中山大学孙逸仙纪念医院 | Culture medium for culturing CD90posi cells and culture method thereof |
| CN112501121A (en) * | 2020-11-24 | 2021-03-16 | 益善生物技术股份有限公司 | Basic culture medium and culture method for circulating tumor cells |
| CN112501121B (en) * | 2020-11-24 | 2023-05-23 | 益善生物技术股份有限公司 | Basic culture medium and culture method for circulating tumor cells |
| CN115125212A (en) * | 2022-06-17 | 2022-09-30 | 成都诺医德医学检验实验室有限公司 | Peripheral blood circulation tumor cell organoid culture method |
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