CN103898058B - A kind of three-dimensional culture method of novel gum knurl stem cell and its application - Google Patents
A kind of three-dimensional culture method of novel gum knurl stem cell and its application Download PDFInfo
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- CN103898058B CN103898058B CN201410129815.XA CN201410129815A CN103898058B CN 103898058 B CN103898058 B CN 103898058B CN 201410129815 A CN201410129815 A CN 201410129815A CN 103898058 B CN103898058 B CN 103898058B
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Abstract
The present invention completes the enrichment culture of glioma stem cells using three-dimensional collagen scaffold under low-serum-concentration, and a kind of new, stem cell culture method and application closer to internal true environment are provided for glioma in vitro test research.From collagen as the material for making 3D supports in the three-dimensional culture method, collagen is the most important composition of extracellular matrix, good adhesion and migration environment can be provided for cell, and there is good biocompatibility, mechanical strength, degradation coefficient and low immunogenicity.Influence of the present invention research porous collagen scaffold three-dimensional culture to glioma cell dryness, inquire into its mechanism, grope the targeting GSCs drug test methods closer to in-vivo tumour stem cell environment, and drug sensitive test is carried out, then glioma resistance mechanism is further studied, new thinking is provided for Ji hair GSCs targeted drugs to lay the foundation, its achievement has certain theory significance and application value.
Description
Technical field
The invention belongs to cell biological field, the three-dimensional culture method of more particularly to a kind of novel gum knurl stem cell and its
Using.
Background technology
Glioblastoma is as incidence of disease highest malignant tumour in nervous system, in past 10 years, even if experience
Operation, the greater advance of chemotherapy and radiotherapy, its median survival interval were also only improved to 14 months by 10 months, and in pin
To in multiple large-scale, polycentric drug discovery assay of glioma, all failing to achieve satisfactory results, most of medicine
The reminder of a fierce battle of a beaten army in phase ii clinical trial, fail to embody the conclusion consistent with during in vitro test.This in vitro test is with facing
The situation that bed result is not consistent generally existing in anti-glioma Therapy study, imply that the morbidity at present for glioblastoma
The understanding of mechanism, developing characteristics and resistant mechanism also has many blank, anti-glioblastoma tumour medicine in vitro test method
There is further improved space.
The discovery of tumor stem cell and progress in recent years, also it is the key such as glioma Preventive and treatment resistance
Mechanism and research and development diagnosis and treatment new technology opens the new visual field on this basis, and increasingly becomes the heat of glioma research field
Point.Glioma stem cells (glioma stem cells, GSCs) are because of its self-renewing, Multidirectional Differentiation and the energy to treatment resistance
Power, it is to maintain malignancy of tumor growth and relapse and metastasis " seed cell ", therefore is controlled by the anti-glioma being predominantly targeting of GSCs
Treat research also as anti-Treatment for Glioma emerging emphasis direction it
The research for so carrying out targetting GSCs treatments requires us to have one to carry out in vitro culture for GSCs first
Technology and carry out being different from traditional materia medica test method on this technology.In the conventional anti-cancer agent research and development of my section
In, we are essentially according to traditional tumour medicine in vitro test method, i.e., next to detect reaction of the single-layer culturing cell to medicine
Carry out.This method is created by National Cancer Institute (NCI) in last century the eighties, but in clinical practice, Chang Fa
The drug effect deviation of the sensitive effect of drugs of existing in vitro test and patient's body solid tumor clinical assessment is larger.Both there are some researches show,
This is at least partly due to that the in-vitro cell growth of monolayer cultivation loses three-D space structure, can not true antimer
Physiopathological structures, growth conditions and the radiation-resistant tumor stem cell level of relative resistance of inner tumour cell.
Into after the GSCs research epoch, GSCs extracorporeal culturing methods the most frequently used at present are by Reynolds in 1992
BA et al. is in the serum free suspension cultivation for from mouse corpus straitum invent in being separately cultured of NSC.The cultural method
The characteristics of be：Serum-free, the nutrient solution for adding high concentration growth factor, and low adhesion culture environment.Its advantage is：Can be with
Self-renewing and differentiation capability fairly simple, that intuitively show individual cells from cell aspect upper body；But defect is also apparent from：Remove
Balling-up culture repeatability is poor, is difficult to observe in cell ball, it is often more important that it is far from each other with vivo environment.It can not
Travel motion, by unicellular division balling-up, the effect of cell and matrix can not be embodied, Long Term Passages success rate is extremely low, and
In-vivo tumour stem cell suspended state absolutely not, it is not movable, its be adhered to support to form by other cells and matrix it is special micro-
Environment, i.e., in specific " stem cell niche ", serum composition can also be touched.
Apply at present includes in the main material for preparing 3D cell culture：1. natural organic matter：Collagen, chitosan, grape
Sugared aminoglycan class, alginates, fibroin, agarose, starch；2. inorganic macromolecule polyalcohol：PGA (polyglycolic acid), PLA
(PLA), PLC (polycaprolactone), POE (poe) and its heterogeneous polymerization support such as PLGA etc..
Therefore, a kind of new glioma stem cells cultural method is continued at present, truly to reflect interior tumor cell
Physiopathological structures, growth conditions and the radiation-resistant tumor stem cell of relative resistance it is horizontal.
The content of the invention
It is an object of the invention to provide a kind of three-dimensional culture method of novel gum knurl stem cell, the three-dimensional culture method
Good adhesion and migration environment can be provided for cell, and with good biocompatibility, mechanical strength, degradation coefficient and low
Immunogenicity.Unnecessary clinical I, II phase will be effectively avoided to test using this method of Three-dimensional cell culture, so as to reduce
The waste of patient's resource and follow-up study fund.
The purpose of the present invention is achieved through the following technical solutions：
A kind of three-dimensional culture method of novel gum knurl stem cell, using three-dimensional collagen scaffold, and find out suitable low
Serum free culture system concentration, the cultural method comprise the following steps：
1) three-dimensional collagen scaffold is cut into 1*5*5mm cube, stayed overnight using preceding Co60 illumination-based disinfections, 4 DEG C of dryings
Ambient storage is standby, before cell to be seeded, with cell culture fluid dipping pretreatment 12 hours；
2) glioma cell line digestion centrifugation is cleaned 2 times to single cell suspension, then with PBS, to remove serum composition, and
Cell count is carried out, with each three-dimensional collagen scaffold 1 × 104~5 × 104Glioma cell is inoculated in step by the density of individual cell
On three-dimensional collagen scaffold after rapid 1) processing, 4 hours are stood, after cell is effectively adhered on support, is placed in Tissue Culture Plate
Middle culture, change 1 nutrient solution within every 2 days, that is, obtain 3D glioma cell models.
Further, the cell culture fluid described in step 1) is the DMEM of 1% hyclone of addition.
Further, the average pore size of the three-dimensional collagen scaffold is 50 μm.
Further, the material of the three-dimensional collagen scaffold is type i collagen.
Further, glioma cell line is U87 cells.
Present invention beneficial effect compared with prior art is：
1st, prepare the main material selection collagen of 3D cell culture in the present invention, collagen be extracellular matrix it is most important into
Point, good adhesion and migration environment can be provided for cell, and with good biocompatibility, mechanical strength, degradation coefficient
And low immunogenicity；
2nd, using Three-dimensional cell culture of the present invention, in the preclinical drug sensitive test and drug screening of rare malignant tumor
In, it will effectively avoid unnecessary clinical I, II phase from testing, so as to reduce the waste of patient's resource and follow-up study fund；
3rd, the glioma cell obtained using the method for the invention culture, can self-assemble in three-dimensional rack microenvironment
Into lumps, its cell growth form and two dimension culture cell are totally different, and the more two-dimentional culture cell of cell membrane surface structure is containing richer
Rich raised sample or cilium spline structure, it is closer with people's tumor growth glioma cell form, it is dry thin beneficial to follow-up study glioma
Born of the same parents' medicine；
4th, the present invention takes the lead in studying the new technology using three-dimensional collagen scaffold as glioma stem cells culture, more preferable analogue body
Interior glioma stem cells microenvironment, provide new experiment in vitro for further research and development targeting glioma stem cells medicine and put down
Platform.
Brief description of the drawings
Fig. 1 is that laser confocal microscope shows cell distribution situation；
Fig. 2 is that ESEM shows cell growth form；
Fig. 3 is Flow cytometry cultivating system inner tumour cell Survival.
Embodiment
Embodiment 1
A kind of three-dimensional culture method of novel gum knurl stem cell, using three-dimensional collagen scaffold, and find out suitable low
Serum free culture system concentration, the cultural method comprise the following steps：
1) three-dimensional collagen scaffold is cut into 1*5*5mm cube, stayed overnight using preceding Co60 illumination-based disinfections, 4 DEG C of dryings
Ambient storage is standby, before cell to be seeded, with cell culture fluid dipping pretreatment 12 hours；
2) glioma cell line digestion centrifugation is cleaned 2 times to single cell suspension, then with PBS, to remove serum composition, and
Cell count is carried out, with each three-dimensional collagen scaffold 1 × 104~5 × 104Glioma cell is inoculated in step by the density of individual cell
On three-dimensional collagen scaffold after rapid 1) processing, 4 hours are stood, after cell is effectively adhered on support, is placed in Tissue Culture Plate
Middle culture, change 1 nutrient solution within every 2 days, that is, obtain 3D glioma cell models.
Further, the cell culture fluid described in step 1) is the DMEM of 1% hyclone of addition.
Further, the aperture of the three-dimensional collagen scaffold is 50 μm.
Further, the material of the three-dimensional collagen scaffold is type i collagen.
Further, glioma cell line is U87 cells.
Embodiment 2
The present embodiment is the preferredization scheme on the basis of embodiment 1, there is provided a kind of novel gum knurl stem cell
Three-dimensional culture method.Part same as Example 1 in the present embodiment, the content that refer to public Ji in embodiment 1 understood,
The content of 1 public Ji of embodiment should also be as the content as the present embodiment, be not repeated description herein.
The three-dimensional culture method comprises the following steps：
After the U87 cell dissociations centrifugation of conventional two dimension culture, single cell suspension is prepared into, is inoculated in about 50 μm of aperture
On collagen scaffold, cultivated under the conditions of 1% hyclone (FBS)+DMEM nutrient solutions.Whether observe cell can be with preferably gluing
It is connected on support collagen stroma and grows, fluorescence microscopy is transferred to the U87 cells propagation feelings of green fluorescent protein (GFP)
Condition, ESEM (SEM) and HE section observation cell growth forms, the existence of Flow cytometry cultivating system inner tumour cell
Situation (AnnexinV+PI methods), laser confocal microscope three-dimensional reconstruction method observation cell distribution situation in support.Referring to figure
1st, Fig. 2.Wherein Fig. 1, A. GFP-transfected U87 cells can on support multiplicative growth；B, C, D. laser confocal microscope show
Show distribution situation of the cell on support, wherein cell distribution is more in the range of 64-125 μm；E. different propagation is shown
Speed, the U87 cell proliferation rates of 3D cultures are slower than 2D monolayer cultivations, and its growth tendency is shallower；F. show in 3D cultures
Apoptosis ratio higher than 2D culture, this also reflects the multiplication characteristic different from 2D cultures.
The Chinese Academy of Sciences of support mountain described in the present embodiment development biology is prepared to be provided, and material is type i collagen.The support please
Referring to document, CHEN L, XIAO Z, MENGL, et a1.The enhancement of cancer stem cell
properties of MCF-7cells in3D collagen scaffolds for modeling of cancer and
Anti-cancer drugs [J] .Biomaterials, 2012,33 (5)：1437-44.
Observe three-dimensional collagen scaffold culture influences on glioma cell dryness：By different modes (two-dimentional vs is three-dimensional) and difference
The cell dissociation of serum condition (10%vs3%vs1%vs0) culture is into after single cell suspension, with Flow cytometry colloid
The ratio of knurl dryness cell (U87CD133+U87 cells).Find out the suitable of three-dimensional collagen scaffold enrichment culture glioma stem cells
Suitable condition, including different serum-concentration nutrient solutions, different time sections collect cell and with suspend into glomus cell compared with etc., herein
The method that three-dimensional collagen scaffold culture glioma stem cells are found on basis.The suitable condition is：Support inner cell is soaked
Not in 1% hyclone+DMEM nutrient solutions, in 37 DEG C, 5%CO2, cultivate in saturated humidity incubator, it is general when collecting cell
Between with 5 days or so.
Glioma U87 cells in three-dimensional collagen scaffold can progressive propagation growth, but the more two-dimentional culture of growth rate is aobvious
Work property slows down, and its difference has conspicuousness, referring to Fig. 1.Glioma cell is further observed in three-dimensional collagen using HE sections and SEM
Growing state can be found in support, glioma cell in three-dimensional rack microenvironment can self-assemble into lumps, the life of its cell
Long form and two dimension culture cell are totally different, and the more two-dimentional culture cell of cell membrane surface structure contains more rich raised sample or cilium sample knot
Structure, it is closer with people's tumor growth glioma cell form, referring to Fig. 2.Dimensional culture is carried out under various concentrations to glioma
The observation that U87 cells dryness influences, progress dimensional culture can substantially carry to glioma cell dryness under 1%FBS concentration conditions
Rise, and stem cell ratio is not weaker than the suspension pelletizing method of classics.Further carry out dryness gene, drug resistant gene RT-PCR and
Western blotting detection, find the Expression of Drug-resistance-associated Genes such as dryness gene and MGMT such as CD133, Sox2, Nanog
Significantly up-regulation, it is especially most notable with Sox2, MGMT up-regulation, referring to Fig. 3.Flow cytometry is shown under 1% serum condition, three-dimensional
In collagen scaffold cultivate glioma stem cells (U87CD133+) ratio up-regulation (A10%FBS, B3%FBS, C1%FBS, D without
FBS), under the conditions of being somebody's turn to do, RT-PCR shows dryness factor gene up-regulated expression (E), and part drug resistant gene raises, and MGMT is most obvious
(F).Western blotting show correlation factor protein level up-regulation (G).Demonstrate 1%FBS+DMEM and utilize three-dimensional collagen
Support culture glioma U87 cells can raise cell dryness, enrichment culture stem cell, the cell cultivated simultaneously drug resistant gene and
Protein upregulation.
Claims (1)
1. a kind of three-dimensional culture method of novel gum knurl stem cell, it is characterised in that utilize three-dimensional collagen scaffold, and find out
Suitable low serum free culture system concentration, the cultural method comprise the following steps：
1) three-dimensional collagen scaffold is cut into 1*5*5mm cube, stayed overnight using preceding Co60 illumination-based disinfections, 4 DEG C of dry environments
Store for future use, before cell to be seeded, with cell culture fluid dipping pretreatment 12 hours；
2) glioma cell line digestion centrifugation is cleaned 2 times to single cell suspension, then with PBS, to remove serum composition, and carried out
Cell count, with each three-dimensional collagen scaffold 1 × 104~5 × 104Glioma cell is inoculated in step 1) by the density of individual cell
On three-dimensional collagen scaffold after processing, 4 hours are stood, after cell is effectively adhered on support, is placed in Tissue Culture Plate and trains
Support, change 1 nutrient solution within every 2 days, that is, obtain 3D glioma cell models；
Cell culture fluid described in step 1) is the DMEM of 1% hyclone of addition；
The average pore size of the three-dimensional collagen scaffold is 50 μm；
The material of the three-dimensional collagen scaffold is type i collagen；
Glioma cell line is U87 cells.
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| CN104232584B (en) * | 2014-09-15 | 2017-05-10 | 中国药科大学 | Building of in-vitro three-dimensional cell model of breast cancer and application of in-vitro three-dimensional cell model in research of drug resistance mechanism and reversal agents screening |
| CN104312975B (en) * | 2014-09-25 | 2018-06-12 | 中国人民解放军第三军医大学第一附属医院 | A kind of external dimensional culture model of glioma stem cells and application |
| CN106434562B (en) * | 2016-09-19 | 2020-03-13 | 广州迈普再生医学科技股份有限公司 | Brain tumor in-vitro model for three-dimensional biological printing and construction method thereof |
| CN107058456A (en) * | 2017-06-02 | 2017-08-18 | 四川精准医学检验有限责任公司 | A kind of circulating tumor cell 3D susceptibility test methods |
| CN107523544B (en) * | 2017-10-16 | 2020-03-31 | 中国人民解放军第三军医大学第一附属医院 | Preparation method and application of tumor cell ball |
| CN110862954A (en) * | 2018-08-28 | 2020-03-06 | 广州洁特生物过滤股份有限公司 | Cell culture method based on three-dimensional cell culture support |
| CN110257335B (en) * | 2019-04-10 | 2020-09-29 | 首都医科大学附属北京天坛医院 | Single-layer or multi-layer 3D glioma cell culture model and construction method and application thereof |
| CN110904044B (en) * | 2019-12-17 | 2022-07-19 | 陕西师范大学 | Three-dimensional culture method of tumor stem cells |
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| A three-dimensional collagen scaffold cell culture system for screening anti-glioma therapeutics;Donglai Lv等;《Oncotarget》;20160728;第7卷(第35期);56904-56914 * |
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