CN106047813A - Rapid extraction method of fibroblast and application - Google Patents
Rapid extraction method of fibroblast and application Download PDFInfo
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- CN106047813A CN106047813A CN201610537670.6A CN201610537670A CN106047813A CN 106047813 A CN106047813 A CN 106047813A CN 201610537670 A CN201610537670 A CN 201610537670A CN 106047813 A CN106047813 A CN 106047813A
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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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- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention discloses a rapid extraction method of fibroblast. The method comprises the following steps that 1, isolated tumor tissue is taken and cut into pieces, and a collagenase solution is added for digestion; 2, the mixture is transferred into a centrifugal tube for oscillation, incubation and centrifuging, and sediments are taken to be placed into a cell incubator for culture; 3, the collagenase solution is added for digestion, and the step 2 is executed again to obtain tumor tissue of the loose organization structure obtained after alternate digestion and culture; 4, a pancreatin solution is added, fetal cattle serum is added after normal temperature standing for terminating digestion, suspended solids are poured away, and attaching-wall tumor single cells obtained after trypsin digestion are obtained; 5, a DMEM complete medium is added for subculture, tumor-associated fibroblast is obtained. The invention further provides application of the extraction method. The fibroblast obtained through the extraction method is high in purity, the fibroblast highly expresses FAP, the expression rate is 99.2%, and the method is suitable for medicine popularization.
Description
Technical field
The invention belongs to biomedical sector, particularly relate to a kind of fibroblastic rapid extracting method and application.
Background technology
Tumor associated fibroblast cell (CAF) is topmost cell component in mesenchyma stroma of tumors, tumor generation and send out
Vital role is play during exhibition.CAF can raise endothelial progenitor cells and promote tumor neogenetic blood vessels to tumor by local
Generation and the growth of tumor.These tumor associated fibroblast cells are except expressing this fibroblastic labelling of vimentin
Beyond the region of objective existence, the most specific expressed FAP, α-SMA, but do not express CK, desmin, form, function, protein expression, to cell because of
In the reactivity of son and genetic level, there is notable difference with normal fibroblast (NF) in it.
Tumor associated fibroblast cell is currently one of study hotspot, and a lot of mechanism remain in dispute, but generally believe
This cell has inseparable relation with occur development and the transfer invasion and attack of tumor.Tumor associated fibroblast cell can be secreted
Change factor 12(CXCL12) and hepatocyte growth factor (HGF), the former can directly stimulate tumor cell to carry out breeding and shifting, after
Person causes the change of extracellular matrix (ECM), in turn results in internal epithelial cell proliferation and vicious transformation occurs.In addition,
CXCL12 and HGF affects tumor neovasculature generation together with the VEGF (VEGF) of tumors secrete.With this
Mode, tumor associated fibroblast cell can affect the paracrine factor of normal epithelium cell and tumor cell to affect tumor simultaneously
Develop, increasing evidence show in tumor stroma fibroblastic sudden change be frequently experienced in cancer development it
Before.Medicine with tumor associated fibroblast cell as target spot is a kind of new method improving tumor cure rate.The most this intrinsic
Difference is the target spot that the design of novel drugs and the high selectivity of therapeutic strategy provide multiple uniqueness.But in existing tumor
In associated fibroblast cell extraction method, it is impossible to obtain substantial amounts of tumor associated fibroblast cell, for studying its mechanism and effect
Bring the biggest impact.
Summary of the invention
It is an object of the invention to: for the problem of above-mentioned existence, it is provided that a kind of fibroblastic rapid extracting method
And application.The extracting method of the present invention can effectively, quickly improve extracted amount and the purity of tumor associated fibroblast cell.
To achieve these goals, the technical solution used in the present invention is as follows:
A kind of fibroblastic rapid extracting method, comprises the following steps:
S1) take in vitro tumor tissues, shred, then digest toward the tumor tissues after shredding adds collagenase solution,
Obtain the tumor tissues after collagenase digesting；
S2), after transferring to the tumor tissues after described collagenase digesting centrifuge tube be carried out shake, hatch and be centrifuged, precipitation is taken
Thing is put in cell culture incubator and is cultivated, and obtains the tumor tissues that organizational structure is loose；
S3) add collagenase solution to digest, and repeat step S2), obtain the organizational structure after alternating digestion, cultivation
Loose tumor tissues；
S4) adding trypsin solution in the loose tumor tissues of described organizational structure, room temperature adds hyclone after standing and terminates
Digestion, then float is outwelled, tumor adherent after obtaining trypsinization is unicellular；
S5) the unicellular middle addition DMEM complete medium of adherent after described trypsinization tumor carries out Secondary Culture, obtains
Described tumor associated fibroblast cell.
Further, in step S1), described in vitro tumor tissues is shredded before, also include in vitro tumor tissues
Soaking 1-3min in PBS, the molar concentration of described PBS is 0.01mol/L, and described collagenase solution is Collagenase I solution.
Further, in step S1), described molar concentration is that the compound method of the PBS of 0.01mol/L is: by KCL
0.2g、KH2PO40.24g, NaCl 8.0g and Na2HPO41.44g is dissolved in 1000mL DDH2In O；Described Collagenase I solution
Quality-the volumetric concentration of middle Collagenase I is 0.8-1.5mg/mL.
Further, in step S2), the frequency of described concussion is 1500-2000rpm, and the time is 2-4min；Described hatch
For hatching 1-3min in 35-38 DEG C of water-bath；Described concussion and hatch alternately 2-4 time.
Further, in step S2), described being centrifuged is centrifuged 5-7 min for the tumor tissues after hatching under 300g, abandons
Supernatant；Adding 18-25 mL molar concentration in precipitation is the PBS of 0.01mol/L, is centrifuged 5-after mixing under 300g
7min, abandons supernatant；Adding 18-25 mL molar concentration again in precipitation is the PBS of 0.01mol/L, after mixing under 300g
Centrifugal 5-7min, abandons supernatant, taking precipitate.
Further, in step S2), CO in described cell culture incubator2Percent by volume be 6-8%, temperature is 35-38
DEG C, the time of cultivation is 1-3 days.
Further, in step S4), the compound method of described trypsin solution is: by NaCl 7-9g, NaHCO3 0.4-
0.6g, glucose 0.8-1.2g, EDTA 0.2-0.4g and pancreatin 2.3-2.8g are dissolved in 1000mL DDH2In O；Described room temperature
The time stood is 1-2min.
Further, in step S5), before carrying out Secondary Culture, also include adherent after described trypsinization with PBS
Tumor unicellular washing 2-3 time；Described DMEM complete medium be in the incomplete culture medium of DMEM add hyclone and
The solution that P/S obtains, wherein the percent by volume of hyclone is 10%, and the percent by volume of P/S is 1%.
Present invention also offers a kind of fibroblastic rapid extracting method and extract tumor associated fibroblast cell
Application.
Further, described tumor is solid tumor, and this solid tumor is pulmonary carcinoma, hepatocarcinoma, intestinal cancer, carcinoma of prostate, cancer of pancreas
With one or more in adenocarcinoma of lung.
In sum, owing to have employed technique scheme, the invention has the beneficial effects as follows: utilize the one-tenth fiber of the present invention
The fibroblast purity that the rapid extracting method of cell obtains is the highest, fibroblast high expressed FAP, and expression rate is
99.2%.Under microscope, tumor associated fibroblast cell is spindle shape or asterism shape, and kytoplasm enriches, and acidophilia, karyon is positioned at cell space
Central authorities, circular or oblong, cell is radially or pencil arranges, and part cell arrangement is disorderly, loss of polarity, has significantly
Juxtaposition phenomenon.Immunocytochemical stain result is pointed out, and the Fibroblast specific binding anti-FAP monoclonal of energy is glimmering
Photoactivated antibody, sends red fluorescence.It is demonstrated experimentally that fibroblastic rapid extracting method of the available present invention extracts tumor phase
Close fibroblast, be suitable for medical science and promote.
Accompanying drawing explanation
Examples of the present invention will be described by way of reference to the accompanying drawings, wherein:
Fig. 1 is to peel off, from tumor-bearing mice, the tumor tissues obtained.
Fig. 2 is the tumor tissues shredded.
Fig. 3 is the cellular morphology under tumor associated fibroblast microcytoscope.
Fig. 4 is the FAP expression rate of the tumor associated fibroblast cell utilizing extracting method of the present invention to obtain.
Fig. 5 is the reality of the immunocytochemical stain of the tumor associated fibroblast cell utilizing extracting method of the present invention to obtain
Test result.
Detailed description of the invention
All features disclosed in this specification, or disclosed all methods or during step, except mutually exclusive spy
Levy and/or beyond step, all can combine by any way.
Any feature disclosed in this specification (including any accessory claim, summary), unless specifically stated otherwise,
By other equivalences or there is the alternative features of similar purpose replaced.I.e., unless specifically stated otherwise, each feature is a series of
An example in equivalence or similar characteristics.Material used in following embodiment, reagent etc., if no special instructions, all
Commercially obtain.
One, the material used in embodiment and reagent
Ophthalmology is cut to mostly apparatus medical treatment company limited product, and model is YYJ-PT ＞ 10cm curved scissors.This eye scissors head end is wide by 0.4
Millimeter, point and the sharpest.
Human hepatoma HepG2 cell (ATCC, article No. HB-8065), the public can obtain this biomaterial at applicant, and this is raw
Thing material only attach most importance to duplicate invention related experiment used by, can not use as other purposes.
BALB/c nude mice (Beijing Vital River Experimental Animals Technology Co., Ltd., article No. 401), the public can be at applicant
Obtain, this biomaterial only attach most importance to duplicate invention related experiment used by, can not use as other purposes.
Oscillator is IKA product, and model is VORTX1.
Hyclone (FBS) is Gibco Products, article No. 10099-141.
Collagenase I solution is to add tire in the incomplete culture medium of DMEM (Gibco Products, article No. 11995-065)
Ox blood serum and P/S(penicillin (Suo Laibao, article No. P8420) and streptomycin (Suo Laibao, article No. S8290-5)) solution that obtains,
Wherein the concentration of volume percent of hyclone is 10%, and the concentration of volume percent of P/S is 1%；The concentration of Collagenase I in P/S
For 100mg/mL.In this Collagenase I solution, the concentration of Collagenase I is 1mg/mL.
Trypsin solution is to DDH2O adds NaCl, NaHCO3, the solution that obtains of glucose, EDTA and pancreatin, wherein,
The concentration of NaCl is 8mg/mL, NaHCO3Concentration be 0.5mg/mL, the concentration of glucose is that the concentration of 1mg/mL, EDTA is
0.3mg/mL, the concentration of pancreatin is 2.5mg/mL.
The compound method of 0.01mol/L PBS is: KCL 0.2g, KH2PO40.24g, NaCl 8.0g, Na2HPO4
1.44g is dissolved in 1000mL DDH2O。
DMEM complete medium is to add hyclone in the incomplete culture medium of DMEM (Gibco, article No. 11995-065)
The solution obtained with P/S(penicillin (Suo Laibao, article No. P8420) and streptomycin (Suo Laibao, article No. S8290-5), wherein tire cattle
The concentration of volume percent of serum is 10%, and the concentration of volume percent of P/S is 1%.
Two, the extraction of tumor associated fibroblast cell
Step S1): take 3 4-5 week old BALB/c nude mices, in oxter, right side subcutaneous vaccination human hepatoma HepG2 cell, wait to swell for every
Tumor average external volume length is to about 1.0 × 1.0 × 1.0cm3Time, obtain tumor-bearing mice.After death soak at tumor-bearing mice cervical dislocation
In 75%(concentration of volume percent) ethanol water in 3-5 min, the mice after being sterilized；In superclean bench, use
Mouse skin is cut off in sterile scissors and aseptic nipper mice chest after sterilization, and careful stripping obtains tumor tissues.Accompanying drawing 1 is
The tumor tissues obtained is peeled off from tumor-bearing mice.
Somewhat shred tumor tissues (tumor tissues size now is 0.5 × 0.5 × 0.5cm with eye scissors3), and
It is drawn off after 0.01mol/L PBS soaks (making tumor tissues complete wetting in 0.01mol/L PBS) 2min, is soaked
Tumor tissues after bubble.(tumor tissues now is big fully to shred the tumor tissues after immersion on sterilized petri dishes with eye scissors
Little is 0.2 × 0.2 × 0.2mm3), drip 0.01mol/L PBS during the tumor tissues after shredding immersion in good time, protect
Hold this tumor tissues and be in moisture state, obtain the tumor tissues shredded.Accompanying drawing 2 obtains for fully shredding tumor tissues with eye scissors
To the tumor tissues shredded.Collagenase I solution is dripped, with cutting in the sterilized petri dishes of the above-mentioned tumor tissues filling and shredding
1 mL rifle head of head dispels tumor tissues, obtains Collagenase I-tumor tissues mixture.
In said method, described eye scissors is cut also known as ophthalmologic operation, ophthalmic operating scissors, is the device shearing eye soft tissue
Tool.Described eye scissors can be that head end is wide 0.4 millimeter, sharp and the sharpest eye scissors；It is wide 1.6 millimeters that described eye scissors is alternatively head end
Eye scissors.
Step S2): Collagenase I-tumor tissues mixture is transferred to, in 50mL centrifuge tube, then be carried out by this centrifuge tube
Following C1) and process C2), C1) and C2) alternately 3 times:
C1) carry out shaking 3 min on the 50mL centrifuge tube earthquake device of Collagenase I-tumor tissues mixture by filling, shaken
Collagenase after swinging-tumor tissues mixture；
C2) collagenase-tumor tissues mixture after concussion is hatched 2 min in 37 DEG C of water-baths, obtain collagenase digesting
After tumor tissues A.
By step C2) collagenase digesting after tumor tissues A be centrifuged process:
C1) tumor tissues after collagenase digesting after hatching is centrifugal 6 min under 300g, abandon supernatant；
C2) to step c1) precipitation in add 20 mL 0.01mol/L PBS, after mixing under 300g centrifugal 6 min, abandon
Clear liquid；
C3) to step c2) precipitation in add 20 mL 0.01mol/L PBS, after mixing under 300g centrifugal 6 min, abandon
Clear liquid, obtains the tumor tissues after collagenase digesting.
Tumor tissues after above-mentioned collagenase digesting is layered in culture bottle, adds DMEM complete medium, train at cell
Support in case and cultivate two days, CO in cell culture incubator2Percent by volume be 6-8%, temperature is 35-38 DEG C, obtains organizational structure
Loose tumor tissues.
Step S3): in loosely organized tumor tissues, add collagenase solution again digest, and repeat step S2),
Obtain the tumor tissues that the organizational structure after alternating digestion, cultivation is loose.
Step S4): in the tumor tissues that organizational structure is loose, add trypsin solution, obtain pancreatin-tumor tissues mixing
Thing, by pancreatin-tumor tissues mixture after room temperature on superclean bench stands 1min, adds 10mL hyclone FBS eventually
Only digestion, outwells pancreatin tumor tissues suspension, and tumor adherent after obtaining trypsinization is unicellular.After trypsinization adherent
The unicellular middle addition DMEM complete medium of tumor carries out Secondary Culture, obtains tumor associated fibroblast cell after purification.
Step S5): being cultivated by above-mentioned tumor associated fibroblast cell, condition of culture is: DMEM complete medium, carefully
Born of the same parents' incubator (CO in cell culture incubator2Percent by volume be 6-8%, temperature is 35-38 DEG C), obtain adherent tumor be correlated with
Fibroblast.Accompanying drawing 3 is the cellular morphology under tumor associated fibroblast microcytoscope.
The fibroblastic rapid extracting method of the present invention can be applicable to extract tumor associated fibroblast cell.Described tumor
For solid tumor, this solid tumor is one or more in pulmonary carcinoma, hepatocarcinoma, intestinal cancer, carcinoma of prostate, cancer of pancreas and adenocarcinoma of lung.Described
Tumor concretely people's hepatocarcinoma of growth on BALB/c nude mice.
Three, the qualification of tumor associated fibroblast cell
1. the expression detection of tumor associated fibroblast cell FAP
Measure tumor associated fibroblast cell with flow cytometer and express the level of FAP.First by tumor associated fibroblast cell
With anti-(0.5g/1 × 106Cell) (Abcam Products, article No. is ab54651) 22 DEG C of reactions in 0.01mol/L PBS
30 minutes, reaction was washed three times with 0.01mol/L PBS after terminating, and two with DyLight 488 labelling resist the most again
(0.01mol/L PBS dilutes, and dilution ratio is 1/500) (Abcam Products, article No. is ab96879) reacts 30 points at 4 DEG C
Clock, reacts and washes three times with 0.01mol/L PBS after terminating, and upper machine analysis, as shown in Figure 4, in Fig. 4, left peak is negative control to result
Peak, right peak is positive findings peak, and expression is the expression of FAP；Result display tumor associated fibroblast cell purity is the highest,
High expressed FAP, expression rate is i.e. expressed the tumor associated fibroblast cell of FAP and is accounted for that to be detected tumor associated fibroblast thin by 99.2%(
The percentage ratio of born of the same parents is 99.2%).
2. tumor associated fibroblast cellular immunization cytochemical staining experiment
(1) by the most adherent tumor associated fibroblast plating cells in 6 orifice plates, with the culture medium culturing 24 without serum
Hour, cell density can be 5 × 105/ hole.
(2) 0.01mol/LPBS washs 2 times, and 4% paraformaldehyde fixes 30min.
(3) with the hyclone that concentration is 5%, non-specific binding is closed, under room temperature, react 30min.
(4) containing anti-FAP monoclonal one anti-(being made into concentration is 1ug/mL), (Abcam Products, article No. is in replacing
Ab28244) 0.01mol/L PBS, 4 DEG C of reactions are overnight.
(5) 0.01mol/L PBS washs 2 times, the anti-(0.01mol/L with the two of AlexaFluor 594 red fluorescence labelling
PBS dilutes, and dilution ratio is 1/400) (Abcam Products, article No. is ab150080) normal-temperature reaction 30 minutes.
(6) 0.01mol/L PBS washs 2 times, and to be made into concentration be 0.14mg/mL to dye DAPI() 3 minutes, 0.01mol/L
PBS washs 2 times, fluorescence microscopy Microscopic observation.
Experimental result sends red fluorescence after being shown as the specific binding anti-FAP monoclonal antibody of fibrocyte.See accompanying drawing 5
For utilizing the experimental result of the immunocytochemical stain of tumor associated fibroblast cell that the extracting method of the present invention obtains.Aobvious
Under micro mirror, tumor associated fibroblast cell is spindle shape or asterism shape, and kytoplasm enriches, acidophilia, and karyon is positioned at cell space central authorities, circle
Shape or oblong, cell is radially or pencil arranges, and part cell arrangement is disorderly, loss of polarity, has and significantly intersects heavily
Folded phenomenon.Immunocytochemical stain result is pointed out, and Fibroblast can resist by specific binding anti-FAP monoclonal fluorescence
Body, sends red fluorescence.
The invention is not limited in aforesaid detailed description of the invention.The present invention expands to any disclose in this manual
New feature or any new combination, and the arbitrary new method that discloses or the step of process or any new combination.
Claims (10)
1. a fibroblastic rapid extracting method, it is characterised in that comprise the following steps:
S1) take in vitro tumor tissues, shred, then digest toward the tumor tissues after shredding adds collagenase solution,
Obtain the tumor tissues after collagenase digesting；
S2), after transferring to the tumor tissues after described collagenase digesting centrifuge tube be carried out shake, hatch and be centrifuged, precipitation is taken
Thing is put in cell culture incubator and is cultivated, and obtains the tumor tissues that organizational structure is loose；
S3) add collagenase solution to digest, and repeat step S2), obtain the organizational structure after alternating digestion, cultivation
Loose tumor tissues；
S4) to step S3) the loose tumor tissues of described organizational structure adds trypsin solution, room temperature adds tire Sanguis Bovis seu Bubali after standing
Terminating clearly digestion, then float outwelled, tumor adherent after obtaining trypsinization is unicellular；
S5) the unicellular middle addition DMEM complete medium of adherent after described trypsinization tumor carries out Secondary Culture, obtains
Described tumor associated fibroblast cell.
Fibroblastic rapid extracting method the most according to claim 1, it is characterised in that in step S1), described general
Before in vitro tumor tissues shreds, also include in PBS, soak in vitro tumor tissues the mole dense of 1-3min, described PBS
Degree is 0.01mol/L, and described collagenase solution is Collagenase I solution.
Fibroblastic rapid extracting method the most according to claim 2, it is characterised in that described molar concentration is
The compound method of the PBS of 0.01mol/L is: by KCL 0.2g, KH2PO40.24g, NaCl 8.0g and Na2HPO41.44g it is molten
Solution is in 1000mL DDH2In O；In described Collagenase I solution, the quality-volumetric concentration of Collagenase I is 0.8-1.5mg/mL.
Fibroblastic rapid extracting method the most according to claim 1, it is characterised in that in step S2), described shake
The frequency swung is 1500-2000rpm, and the time is 2-4min；Described hatch as hatching 1-3min in 35-38 DEG C of water-bath；Institute
State concussion and hatch alternately 2-4 time.
Fibroblastic rapid extracting method the most according to claim 1, it is characterised in that in step S2), described from
The heart is the centrifugal 5-7 min under 300g of the tumor tissues after hatching, and abandons supernatant；18-25 mL mole is added in precipitation
Concentration is the PBS of 0.01mol/L, is centrifuged 5-7min, abandons supernatant after mixing under 300g；18-25 is added again in precipitation
ML molar concentration is the PBS of 0.01mol/L, is centrifuged 5-7min, abandons supernatant, taking precipitate after mixing under 300g.
Fibroblastic rapid extracting method the most according to claim 1, it is characterised in that in step S2), described carefully
CO in born of the same parents' incubator2Percent by volume be 6-8%, temperature is 35-38 DEG C, and the time of cultivation is 1-3 days.
Fibroblastic rapid extracting method the most according to claim 1, it is characterised in that in step S4), described pancreas
The compound method of enzymatic solution is: by NaCl 7-9g, NaHCO3 0.4-0.6g, glucose 0.8-1.2g, EDTA 0.2-0.4g and
Pancreatin 2.3-2.8g is dissolved in 1000mL DDH2In O；The time that described room temperature stands is 1-2min.
Fibroblastic rapid extracting method the most according to claim 1, it is characterised in that in step S5), carrying out
Before Secondary Culture, also include unicellular for tumor adherent after described trypsinization washing 2-3 time with PBS；Described DMEM is complete
Full culture medium is the solution that addition hyclone and P/S obtain in the incomplete culture medium of DMEM, the wherein volume of hyclone
Percentage ratio is 10%, and the percent by volume of P/S is 1%.
9. a fibroblastic rapid extracting method is in the application extracting tumor associated fibroblast cell.
Application the most according to claim 9, it is characterised in that described tumor is entity tumor, this entity tumor is lung
One or more in cancer, hepatocarcinoma, intestinal cancer, carcinoma of prostate, cancer of pancreas and adenocarcinoma of lung.
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| CN108070561A (en) * | 2016-11-15 | 2018-05-25 | 江苏齐氏生物科技有限公司 | A kind of isolation and culture method of the primary colon cancer cell of people |
| CN109112192A (en) * | 2018-06-28 | 2019-01-01 | 广西医科大学 | A kind of method of unicellular sequencing screening fibroblast mark molecule |
| CN110452878A (en) * | 2018-05-07 | 2019-11-15 | 北京吉尚立德生物科技有限公司 | A kind of lung cancer solid tumor mass sample dissociation solution |
| CN111117965A (en) * | 2020-01-08 | 2020-05-08 | 宁波市医疗中心李惠利医院 | Rapid purification method of high-purity primary tumor cells |
| CN114231479A (en) * | 2021-11-09 | 2022-03-25 | 中国医科大学附属第四医院 | Kit for extracting human primary liver cancer fibroblast and extraction method |
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| CN111117965A (en) * | 2020-01-08 | 2020-05-08 | 宁波市医疗中心李惠利医院 | Rapid purification method of high-purity primary tumor cells |
| CN114231479A (en) * | 2021-11-09 | 2022-03-25 | 中国医科大学附属第四医院 | Kit for extracting human primary liver cancer fibroblast and extraction method |
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