CN109609461A - A kind of primary tumor cell isolation and culture method - Google Patents
A kind of primary tumor cell isolation and culture method Download PDFInfo
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- 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
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Abstract
一种原代肿瘤细胞分离培养方法，包括如下步骤：将肿瘤标本去除肿瘤组织周围的坏死及非肿瘤组织，剪碎、消化，所得消化后组织经筛网过滤、离心后PBS重悬，再经离心，弃上层清液，得分离后的组织细胞；将分离后的组织细胞用原代细胞无血清培养基重悬得到重悬液，调整细胞密度，接种于原代细胞超低粘附培养皿中培养；每隔48小时，离心收集细胞后弃掉原有的原代细胞无血清培养基上清，再用原代细胞无血清培养基重悬换液，继续在原代细胞超低粘附培养皿中培养至1周以上，再经常规细胞培养方法培养得到高纯度的原代肿瘤细胞。本发明制备的细胞获得率高，纯化程度高，且对现有实验设备没有更高的要求，适宜在肿瘤研究领域广泛应用推广。A method for separating and culturing primary tumor cells, comprising the following steps: removing necrotic and non-tumor tissues around the tumor tissue from a tumor specimen, shredding and digesting the obtained tissue, filtering the obtained digested tissue through a mesh, centrifuging, and resuspending in PBS, and then resuspending it in PBS. Centrifuge, discard the supernatant to obtain the separated tissue cells; resuspend the separated tissue cells with the primary cell serum-free medium to obtain a resuspended liquid, adjust the cell density, and inoculate the primary cell ultra-low adhesion culture dish cultured in medium; every 48 hours, the cells were collected by centrifugation and the original supernatant of the primary cell serum-free medium was discarded. The cells were cultured in a dish for more than 1 week, and then cultured by conventional cell culture methods to obtain high-purity primary tumor cells. The cells prepared by the invention have high obtaining rate and high purification degree, and have no higher requirements on the existing experimental equipment, and are suitable for wide application and promotion in the field of tumor research.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of primary tumor cell isolated culture method.
Background technique
Tumor invasion and metabasis etc. is the process an of multi-step, multifactor, continuous progressivity.Growth, evolution in tumour
In the process, due to itself unstability of heredity and the selection pressure of internal external environment, cell will appear continuous variation, lead to it
The diversification of phenotype, and a solid tumor tissue or cell line are exactly the cell subsets institute group different by these biological characters
At.Tumour is mainly shown as in the heterogeneity in terms of characters such as invasion transfer, in the cell colony of a malignant tumour, not
All tumour cells all have invasion and transfer ability, and only certain special cell subsets just have this potential, Er Qie
These have in the cell subsets of potential such as invasion transfer, and the abilities such as their own invasion transfer are also inconsistent.From tumor group
Corresponding tumor cell line is established in knitting, to dependency basis such as the molecular mechanisms and invasion transfer for finding the characters such as decision invasion transfer
Because etc. be of great significance.
It will be appreciated that there is the case where non-tumor cell pollution in existing Tumor cell, such as
It is whole during tumour originally culture to use culture medium+fetal calf serum training mode, under the training mode, tumour cell
And non-tumor cell can be quickly proliferated, even if using the cell of density-gradient centrifugation method separation different densities, but still
It is so difficult to remove non-tumor cell such as fibroblast, it is low to finally result in the tumour cell purity for obtaining and purifying.Due to non-swollen
The primary tumor cell of the presence of oncocyte, acquisition is used in cellular biology of tumor and molecular biology experiment, result
There may be bias, reduce the reliability of experimental result and repeatability, influence follow-up study.
Summary of the invention
The purpose of the present invention is to provide a kind of primary tumor cell isolated culture methods, solve people's primary tumor cell
Microbial recovery rate is lower, the problems such as purity is low, the primary tumor cell pick-up rate of preparation is high, and degree of purification is high, and to existing
Experimental facilities does not have higher requirement, is suitable for being widely applied to promote in tumor research field.
In order to achieve the above objectives, the technical scheme is that
The characteristics of present invention can not be survived by non-tumor cell under serum-free culturing conditions establishes a kind of new point
From, purifying primary tumor cell method, it is efficient that this method, which can be widely applied to various tissue-derived tumour cultures,
The primary tumor cell for obtaining purifying provides new technological means.
The ultralow adherency culture dish of a kind of primary cell for primitive cell culture of the present invention, paved method is such as
Under:
(1) by polymethylacrylic acid 2- hydroxyl second rouge (Poly (2-hydroxyethyl methacrylate, Poly-HEMA)
Be dissolved in straight alcohol and obtain Poly-HEMA glue, wherein in the Poly-HEMA glue Poly-HEMA final concentration >=20mg/
mL；
(2) configured Poly-HEMA glue is covered in culture dish bottom with pipettor, blots residual gum, be placed in thin
Born of the same parents cultivate ultraviolet irradiation in super-clean bench, and blowing is overnight to being completely dried；The culture dish prepared is received into standby in sterile chamber
With.
Preferably, in the Poly-HEMA glue Poly-HEMA final concentration of 20~100mg/mL.
It is furthermore preferred that in the Poly-HEMA glue Poly-HEMA final concentration of 20mg/mL.
Polymethylacrylic acid 2- hydroxyl second rouge (Poly-HEMA) used in the present invention can be commercially available by commercially available mode, example
Such as Sigma Products (25249-16-5).
The present invention also provides a kind of primary cell serum free medium, the preparation methods of the primary cell serum free medium
Are as follows: insulin-like growth factor (IGF), basic fibroblast growth factor are added into basal medium DMEM/F12
(bFGF), epidermal growth factor (EGF), final concentration of 20ng/mL of the primitive cell culture base containing IGF, bFGF final concentration
For 20ng/mL, the final concentration of 20ng/mL of EGF.
Primary tumor cell isolated culture method of the present invention, includes the following steps:
1) tissue treatment, digestion, separation
The tumor specimen trimming of operation excision is removed into necrosis and nonneoplastic tissue around tumor tissues, shreds, digest,
Tissue PBS after metallic sieve filtering, centrifugation is resuspended after gained digestion, then is centrifuged, and supernatant liquor, the tissue after must separating are abandoned
Cell；
2) the ultralow adherency culture of serum-free
Histocyte after separation is resuspended to obtain re-suspension liquid with primary cell serum free medium, adjusts cell density,
With 1 × 106A/L concentration is inoculated in the paved ultralow adherency culture dish of primary cell of the present invention and cultivates；Every 48 hours, from
The heart discards original primary cell serum free medium supernatant after collecting cell, then is changed with the resuspension of primary cell serum free medium
Liquid continues culture in the ultralow adherency culture dish of the primary cell and obtained the primary cell ball of free serum culture to 1 week or more.
3) it is expanded according to regular growth cultural method, obtains the primary tumor cell of high-purity.
Further, the method for step c) amplification are as follows: cell is collected by centrifugation in the primary cell ball of step b) free serum culture,
The tryptic digestive juice containing 0.25%EDTA is added and is digested to individual cells, places in ordinary cells culture bottle according to traditional
Cell culture processes culture.
Again, the primary cell serum free medium is that insulin-like growth factor is added into basal medium DMEM/F12
Sub (IGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) configure, the primary cell training
Support final concentration of 20ng/mL of the base containing IGF, the final concentration of 20ng/mL of bFGF final concentration of 20ng/mL, EGF.
The present invention uses the paved originally culture ware of Poly-HEMA glue, wherein final concentration >=20mg/mL of Poly-HEMA,
Poly-HEMA is a kind of high molecular surfactant-acrylic polymer, has excellent film forming, dispersion, emulsifying capacity.
It is solid powder under normal circumstances, and solid Poly-HEMA is dissolved in ethyl alcohol by the present invention, and is layered in culture dish, when
After ethyl alcohol volatilization, Poly-HEMA can form gel in culture dish bottom, and cell is easy to adherent bottom and is completely cut off, is caused thin
Born of the same parents can not adhere to adherent growth, can be only formed the tumour cell sphere of floating.The final concentration that wherein Poly-HEMA is dissolved in ethyl alcohol is answered
Control >=20mg/mL, preferably in 20~100mg/mL, final concentration cell can occur because gel strength is too low lower than 20mg/mL
Adherent growth can still be adhered to；Final concentration, which then will lead to Poly-HEMA powder higher than 100mg/mL, can not be dissolved completely in ethyl alcohol
In, while excessive Poly-HEMA is consumed, increase bed board cost.
It is thin that culture method in serum-free provided by the invention can effectively remove easily mixed non-tumour in Tumor cell
Born of the same parents' ingredient such as fibroblast, significantly improves tumour cell microbial recovery rate and purity.
Compared with prior art, beneficial effects of the present invention are as follows:
The invention utilizes non-tumor cell can not long-term surviving under serum-free and ultralow adherency training mode
This characteristic isolates and purifies tumour cell.The first step of the present invention is viscous first with primary cell serum free medium+ultralow
Attached culture dish provides serum-free+ultralow adherency training mode, dexterously removes the non-tumor cells such as fibroblast, second step
Routine culture mode (adherent+serum) is taken to expand the tumour cell to survive, it is pure so as to efficiently obtain
The primary tumor cell of change, and cell passage can be carried out, freeze, recover.
Tumour primary separation and culture method provided by the invention is verified in nearly hundred tumour originally cultures reliable
Effectively, and this method is at home and abroad not reported at present.
Detailed description of the invention
Fig. 1 is the primary osteosarcoma cell inverted phase contrast microscope photo that the embodiment of the present invention 3 is cultivated.
Fig. 2 is the primary osteosarcoma cell growth curve that the embodiment of the present invention 3 is cultivated.
Fig. 3 is the primary osteosarcoma cell inverted phase contrast microscope photo that the embodiment of the present invention 4 is cultivated.
Fig. 4 is the primary osteosarcoma cell growth curve that the embodiment of the present invention 4 is cultivated.
Fig. 5 is the primary osteosarcoma cell inverted phase contrast microscope photo that the embodiment of the present invention 5 is cultivated.
Fig. 6 is the primary osteosarcoma cell growth curve that the embodiment of the present invention 5 is cultivated.
Specific embodiment
Now in conjunction with embodiment and attached drawing, the invention will be further described, but implementation of the invention is not limited to that.
Method therefor is conventional method to following embodiments unless otherwise specified.
The reagent used in this experiment is as follows: DMEM/F12, DMEM, FBS are Gibco Products；IGF, bFGF and EGF
For PeproTech Products；Poly-HEMA, 0.25% pancreatin of clostridiopetidase A are Sigma Products；TRIZOL is
Invitrogen Products；PBS configuration method: 8.0g NaCl, 0.2g KCl, 0.2g KH2PO4、1.44g Na2HPO4·
12H2O is dissolved in 1000mL deionized water, and (no special to point out, PBS used in embodiment all matches adjustment pH value to 7.35 thus
Side).
The preparation of 1 primitive cell culture base of embodiment
In superclean bench into basal medium DMEM/F12 be added insulin-like growth factor (IGF), alkalinity at
Fibroblast growth factor (bFGF), epidermal growth factor (EGF), configuration are free of the primitive cell culture base of serum, the culture
The final concentration of 20ng/ml of the base final concentration of 20ng/ml containing IGF, bFGF final concentration of 20ng/ml, EGF.
2 originally culture ware of embodiment it is paved
Polymethylacrylic acid 2- hydroxyl second rouge (Poly-HEMA) is dissolved in straight alcohol, final concentration of 20mg/ml；With shifting
Configured Poly-HEMA glue is covered in culture dish bottom by liquid device, blots residual gum, is placed in cell culture super-clean bench purple
External exposure, blowing is overnight to being completely dried (12 hours).The culture dish prepared is received into spare in sterile chamber.
The highly purified osteosarcoma primary cell of embodiment 3 is separately cultured
Experimental method:
(1) the osteosarcoma tumor sample by operation excision is put into 1000U/mL containing penicillin, 1000 μ g/mL's of streptomysin
It in Hanks liquid, is placed in 50mL centrifuge tube, external ice bag is sent rapidly to laboratory.
(2) tumor tissues are put into 10cm plate, the downright bad and non-tumor group around conventional trimming removal tumor tissues
It knits, physiological saline repeated flushing, antiseptic sursery, which is cut, shreds tissue, is placed in liquid containing 10%DMEM, is added by nutrient solution volume
II Collagenase Type 10mg/mL is placed in 37 DEG C of constant temperature oscillation casees with 150 revs/min of oscillation 2h.
(3) postdigestive tissue specimen is taken out, after the sieving of 300 mesh metallic sieves, the liquid that sieve is already expired slowly is infused
In the centrifuge tube for entering to preset Ficoll lymph separating liquid, 2500 turns of centrifugation 30min.
(4) intermediate layer cell is collected, 1000 turns of centrifugation 5min after PBS is resuspended abandon supernatant, configured in embodiment 1
Primary cell serum free medium be resuspended after, adjust cell density, with 106Density is inoculated in the original prepared in embodiment 2
Contain 5%CO in the ultralow adherency culture dish of cell, being put into 37 DEG C2It is cultivated in the cell incubator of 95% air.
(5) cellular morphology in routine observation culture dish discarded original culture after cell is collected by centrifugation every 48 hours
Base supernatant.Liquid is changed with primitive cell culture base weight configured in embodiment 1 is outstanding again, continues the original prepared in example 2
For culture in Tissue Culture Dish to 1 week or more.
(6) cell is collected by centrifugation after culture 1 week in the primary cell ball of above-mentioned free serum culture, is added and contains 0.25%EDTA
Tryptic digestive juice be digested to individual cells, place and trained in ordinary cells culture bottle according to traditional cell culture mode
It supports, culture medium is changed to DMEM, and 10% fetal calf serum is added in the medium, is put into 37 DEG C containing 5%CO2Cell training
It supports and is cultivated in case, and change liquid according to the passage of regular growth cultural method, obtain highly purified cells of tumorous bone.
Experimental result:
The non-tumor cells such as fibroblast can be effectively removed according to the method described above, can obtain highly purified bone tumour
Cell, and cell passage can be carried out, freeze, recover.
Fig. 1 is the primary osteosarcoma cell form that the present embodiment is separately cultured under light microscopic, as shown in Figure 1, the cell of culture
In polygonal, core is big, and kernel is obvious, meets the morphological feature under tumour cell optical microscopy.
Fig. 2 is the primary osteosarcoma cell growth curve that the present embodiment is separately cultured, as shown in Figure 2, the 1st, 2 day OD450
For value less than 0.5, the 5th day OD450 value is more than 1, illustrates that the cell has quick growing multiplication ability, meets the life of tumour cell
Long feature.
The highly purified thyroid cancer primary cell of embodiment 4 is separately cultured
Experimental method:
(1) the thyroid cancer tumor specimen by operation excision is put into 1000U/mL containing penicillin, 1000 μ g/mL's of streptomysin
It in Hanks liquid, is placed in 50mL centrifuge tube, external ice bag is sent rapidly to laboratory.
(2) tumor tissues are put into 10cm plate, the downright bad and non-tumor group around conventional trimming removal tumor tissues
It knits, physiological saline repeated flushing, antiseptic sursery, which is cut, shreds tissue, is placed in liquid containing 10%DMEM, is added by nutrient solution volume
II Collagenase Type 10mg/mL is placed in 37 DEG C of constant temperature oscillation casees with 150 revs/min of oscillation 2h.
(3) postdigestive tissue specimen is taken out, after the sieving of 300 mesh metallic sieves, the liquid that sieve is already expired slowly is infused
In the centrifuge tube for entering to preset Ficoll lymph separating liquid, 2500 turns of centrifugation 30min.
(4) intermediate layer cell is collected, 1000 turns of centrifugation 5min after PBS is resuspended abandon supernatant, configured in embodiment 1
Primary cell serum free medium be resuspended after, adjust cell density, with 106Density is inoculated in the original prepared in embodiment 2
Contain 5%CO in the ultralow adherency culture dish of cell, being put into 37 DEG C2It is cultivated in the cell incubator of 95% air.
(5) cellular morphology in routine observation culture dish discarded original culture after cell is collected by centrifugation every 48 hours
Base supernatant.Liquid is changed with primitive cell culture base weight configured in embodiment 1 is outstanding again, continues the original prepared in example 2
For culture in Tissue Culture Dish to 1 week or more.
(6) cell is collected by centrifugation after culture 1 week in the primary cell ball of above-mentioned free serum culture, is added and contains 0.25%EDTA
Tryptic digestive juice be digested to individual cells, place and trained in ordinary cells culture bottle according to traditional cell culture mode
It supports, culture medium is changed to DMEM, and 10% fetal calf serum is added in the medium, is put into 37 DEG C containing 5%CO2Cell training
It supports and is cultivated in case, and change liquid according to the passage of regular growth cultural method.
Experimental result:
The non-tumor cells such as fibroblast can be effectively removed according to the method described above, can obtain highly purified bone tumour
Cell, and cell passage can be carried out, freeze, recover.
Fig. 3 is the thyroid carcinoma cell form that the present embodiment is separately cultured under light microscopic, from the figure 3, it may be seen that the cell of culture is in
Polygonal, core is big, and kernel is obvious, meets the morphological feature under tumour cell optical microscopy.
Fig. 4 is the thyroid carcinoma cell growth curve that the present embodiment is separately cultured, as shown in Figure 4, the 1st, 2 day OD450 value
Less than 0.5, the 5th day OD450 value is more than 1, illustrates that the cell has quick growing multiplication ability, meets the growth of tumour cell
Feature.
The highly purified lung cancer cell of embodiment 5 is separately cultured
Experimental method:
(1) the lung cancer tumor sample by operation excision is put into 1000U/mL containing penicillin, 1000 μ g/mL's of streptomysin
It in Hanks liquid, is placed in 50mL centrifuge tube, external ice bag is sent rapidly to laboratory.
(2) tumor tissues are put into 10cm plate, the downright bad and non-tumor group around conventional trimming removal tumor tissues
It knits, physiological saline repeated flushing, antiseptic sursery, which is cut, shreds tissue, is placed in liquid containing 10%DMEM, is added by nutrient solution volume
II Collagenase Type 10mg/mL is placed in 37 DEG C of constant temperature oscillation casees with 150 revs/min of oscillation 2h.
(3) postdigestive tissue specimen is taken out, after the sieving of 300 mesh metallic sieves, the liquid that sieve is already expired slowly is infused
In the centrifuge tube for entering to preset Ficoll lymph separating liquid, 2500 turns of centrifugation 30min.
(4) intermediate layer cell is collected, 1000 turns of centrifugation 5min after PBS is resuspended abandon supernatant, configured in embodiment 1
Primary cell serum free medium be resuspended after, adjust cell density, with 106Density is inoculated in the original prepared in embodiment 2
Contain 5%CO in the ultralow adherency culture dish of cell, being put into 37 DEG C2It is cultivated in the cell incubator of 95% air.
(5) cellular morphology in routine observation culture dish discarded original culture after cell is collected by centrifugation every 48 hours
Base supernatant.Liquid is changed with primitive cell culture base weight configured in embodiment 1 is outstanding again, continues the original prepared in example 2
For culture in Tissue Culture Dish to 1 week or more.
(6) cell is collected by centrifugation after culture 1 week in the primary cell ball of above-mentioned free serum culture, is added and contains 0.25%EDTA
Tryptic digestive juice be digested to individual cells, place and trained in ordinary cells culture bottle according to traditional cell culture mode
It supports, culture medium is changed to DMEM, and 10% fetal calf serum is added in the medium, is put into 37 DEG C containing 5%CO2Cell training
It supports and is cultivated in case, and change liquid according to the passage of regular growth cultural method.
Experimental result:
The non-tumor cells such as fibroblast can be effectively removed according to the method described above, can obtain highly purified bone tumour
Cell, and cell passage can be carried out, freeze, recover.
Fig. 5 is the primary lung carcinoma cell form that the present embodiment is separately cultured under light microscopic, and as shown in Figure 5, the cell of culture is in
Polygonal, core is big, and kernel is obvious, meets the morphological feature under tumour cell optical microscopy.
Fig. 6 is the primary lung cancer cell growth curve that the present embodiment is separately cultured, it will be appreciated from fig. 6 that the 1st, 2 day OD450 value
Less than 0.5, the 5th day OD450 value illustrates that the cell has quick growing multiplication ability, meets tumour cell 1.5 or so
Growth characteristics.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, various changes and improvements may be made to the invention without departing from the spirit and scope of the present invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent defines.
Claims (9)
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110029089A (en) * | 2019-04-29 | 2019-07-19 | 北京和合医学诊断技术股份有限公司 | Serum free medium, preparation method and the method for cultivating primary tumor cell |
| CN111621478A (en) * | 2019-11-05 | 2020-09-04 | 北京基石生命科技有限公司 | Culture method of gynecological tumor primary cells |
| CN111808817A (en) * | 2020-07-24 | 2020-10-23 | 上海昊佰生物科技有限公司 | Culture method of osteosarcoma organs and bone tumor culture medium thereof |
| CN112760283A (en) * | 2019-11-04 | 2021-05-07 | 北京基石生命科技有限公司 | Culture medium for culturing bone and soft tissue tumor solid tumor primary cells |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103352027A (en) * | 2013-05-07 | 2013-10-16 | 中国人民解放军第二军医大学 | Tumor stem cell suspension culture method |
| CN103865876A (en) * | 2014-03-26 | 2014-06-18 | 西北民族大学 | Method for primary culture of tumor cells |
-
2018
- 2018-12-26 CN CN201811600354.4A patent/CN109609461A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103352027A (en) * | 2013-05-07 | 2013-10-16 | 中国人民解放军第二军医大学 | Tumor stem cell suspension culture method |
| CN103865876A (en) * | 2014-03-26 | 2014-06-18 | 西北民族大学 | Method for primary culture of tumor cells |
Non-Patent Citations (3)
| Title |
|---|
| 张伟等: "两种人抗失巢凋亡骨肉瘤细胞模型建立方法的比较", 《局解手术学杂志》 * |
| 颜秉阳等: "肺腺癌胸水肿瘤细胞分离与体外悬浮培养及鉴定", 《现代肿瘤医学》 * |
| 马俊明等: "人肝癌组织中肝癌干细胞的分离培养与初步鉴定", 《宁夏医科大学学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110029089A (en) * | 2019-04-29 | 2019-07-19 | 北京和合医学诊断技术股份有限公司 | Serum free medium, preparation method and the method for cultivating primary tumor cell |
| CN112760283A (en) * | 2019-11-04 | 2021-05-07 | 北京基石生命科技有限公司 | Culture medium for culturing bone and soft tissue tumor solid tumor primary cells |
| CN112760283B (en) * | 2019-11-04 | 2023-05-02 | 北京基石生命科技有限公司 | Culture medium for culturing bone and soft tissue tumor solid tumor primary cells |
| CN111621478A (en) * | 2019-11-05 | 2020-09-04 | 北京基石生命科技有限公司 | Culture method of gynecological tumor primary cells |
| CN111808817A (en) * | 2020-07-24 | 2020-10-23 | 上海昊佰生物科技有限公司 | Culture method of osteosarcoma organs and bone tumor culture medium thereof |
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