CN110129256A - The method for building up of one boar source 3D placental organ model - Google Patents
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- CN110129256A CN110129256A CN201910484492.9A CN201910484492A CN110129256A CN 110129256 A CN110129256 A CN 110129256A CN 201910484492 A CN201910484492 A CN 201910484492A CN 110129256 A CN110129256 A CN 110129256A
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- 230000003169 placental effect Effects 0.000 title claims abstract description 24
- 238000000034 method Methods 0.000 title claims abstract description 15
- 210000000056 organ Anatomy 0.000 title claims abstract description 15
- 210000004027 cell Anatomy 0.000 claims abstract description 48
- 210000002826 placenta Anatomy 0.000 claims abstract description 34
- 210000002220 organoid Anatomy 0.000 claims abstract description 21
- 210000000130 stem cell Anatomy 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims description 20
- 239000006285 cell suspension Substances 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 19
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 12
- 238000005119 centrifugation Methods 0.000 claims description 12
- 210000004698 lymphocyte Anatomy 0.000 claims description 12
- 238000000926 separation method Methods 0.000 claims description 12
- 230000002255 enzymatic effect Effects 0.000 claims description 10
- 239000007864 aqueous solution Substances 0.000 claims description 8
- 230000003115 biocidal effect Effects 0.000 claims description 8
- 230000001413 cellular effect Effects 0.000 claims description 8
- 230000029087 digestion Effects 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 6
- 108010035532 Collagen Proteins 0.000 claims description 4
- 102000008186 Collagen Human genes 0.000 claims description 4
- 102000012422 Collagen Type I Human genes 0.000 claims description 4
- 108010022452 Collagen Type I Proteins 0.000 claims description 4
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 4
- 210000004252 chorionic villi Anatomy 0.000 claims description 4
- 229920001436 collagen Polymers 0.000 claims description 4
- 229940096422 collagen type i Drugs 0.000 claims description 4
- 229910052802 copper Inorganic materials 0.000 claims description 4
- 239000010949 copper Substances 0.000 claims description 4
- 238000011010 flushing procedure Methods 0.000 claims description 4
- 239000002504 physiological saline solution Substances 0.000 claims description 4
- 210000005059 placental tissue Anatomy 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 102000004142 Trypsin Human genes 0.000 claims description 3
- 108090000631 Trypsin Proteins 0.000 claims description 3
- 239000012588 trypsin Substances 0.000 claims description 3
- 230000003196 chaotropic effect Effects 0.000 claims 1
- 239000000725 suspension Substances 0.000 claims 1
- 238000011160 research Methods 0.000 abstract description 11
- 230000007774 longterm Effects 0.000 abstract description 7
- 108010082117 matrigel Proteins 0.000 abstract description 3
- 239000003102 growth factor Substances 0.000 abstract description 2
- 230000006870 function Effects 0.000 description 9
- 239000010410 layer Substances 0.000 description 9
- 210000002993 trophoblast Anatomy 0.000 description 9
- 230000012010 growth Effects 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 3
- 229940088597 hormone Drugs 0.000 description 3
- 239000005556 hormone Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 230000035935 pregnancy Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000007334 copolymerization reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
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- 230000003287 optical effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000021368 organ growth Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000028742 placenta development Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 235000015277 pork Nutrition 0.000 description 1
- 238000009597 pregnancy test Methods 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
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- C12N2513/00—3D culture
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
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Abstract
The invention discloses the method for building up of a boar source 3D placental organ model.It including separating placenta stem-cell, is wrapped in matrigel, organoid growth factor is added, be put into cell incubator culture.It can not long-term cultivation and the shortcomings that passage the present invention overcomes cell line model and primary cell model single cell type, immortalization or cancerization feature, primary cell, can permanently be passed on, be frozen with technical measure culture 3D pig source placenta organoid, present invention firstly provides 3D pig placenta organoid will to basic research and it is applied research have important value.
Description
Technical field
The invention belongs to biological medicines and agricultural biological technical field, more particularly, to a kind of external biological model, specifically
It is the method for building up of a boar source 3D placental organ model.
Background technique
Placenta includes the cell of different function, and different cells cooperate with interaction, the common function of maintaining placenta.Previous research
In, cell line perhaps primary cell cell line mostly immortalization or cancer cell is used more to the research of placental function, therefore,
It is larger with internal normal cell difference, can not true simulation inner body placenta feature and function.However placental trophoblasts
There are many disadvantages for system: such as single cell type, two-dimensional growth, easily morphing.Although primary placenta cells derive from
Internal placenta tissue, but the culture model of its single cell type feature, 2D and can not limit it the disadvantages of long-term cultivation and answer
With.
Since separation placenta tissue is more difficult, and it is related to ethics problem.So the research to placental function still is limited to
Cell level.In current research, researcher utilizes the villus (a kind of small foliation structure) from placenta tissue, these
Trophoderm organoid can long-term surviving, there is genetic stability and be organized into down-like structure, secrete required protein and
Hormone, these protein and hormone will affect metabolism of the mother during pregnancy；It is further analysis shows, organoid with
Normal placenta of early pregnancy is closely similar.Therefore researcher can record positive reaction in pregnancy tests in vitro
The .Trophoblast such as (Turco MY, ' organoids as a model for maternal-fetal
Interactions during human placentation.Nature, 2018,564,263-267).3D placenta organoid
It include cytotrophoblast and plasmoditropho blast functional area, the invention can ensure that 3D pig placental from placenta stem-cell
Organ infinitely passes on, and keeps feature (plasmoditropho blast and cytotrophoblast functional area) constant, therefore the model has
Huge basic research and application study value.
And for a long time, body outer cell line is used for the model of pig evaluation of nutrition.But two-dimentional (2D) cell line model exists
Many inherent defects: being in monolayer growth, can not three-dimensional structure in analogue body；Only contain single cell type, with internal group
It knits and differs greatly with organ many cells composition；Immortalize or cancerization cell line easily morph, with internal cell physiological difference compared with
Greatly；Primary cell can not long-term cultivation in vitro.Therefore, cell line and primary cell model are not ideal external models.Three
Dimension placenta organoid provides possibility to solve this problem.Placental trophoblast organoid derives from placenta stem-cell, in three-dimensional
Three-dimensional growth；The nearly all cell type of placental epithelial in occlusion body；Include placental epithelial villus and crypts functional areas；It can be long-term
Culture is without genetic mutation, is the ideal model for studying placenta growth development, physiology and function therefore, is also research tire
The transhipment of disk nutriment and metabolism provide Important Platform.
China is important pig raising big country, and pork yield and consumption figure account for more than half of world standard.Sow breeding
Degraded performance, the piglet death rate are higher, are still the major obstacle for perplexing the development of China's pig breeding industry.Placental function is that influence sow is numerous
Grow the key of performance and piglet health.Research without pig placenta organoid and report so far.
Summary of the invention
The object of the present invention is to provide the method for building up of a boar source 3D placental organ model, mainly 3D pig tire
Disk trophoderm organoid condition of culture.
The technical scheme of the present invention is realized as follows:
The method for building up of one boar source 3D placental organ model, which is characterized in that its step are as follows:
(1) the isotonic 30% and isotonic 60%Percoll of two parts of 3ml is respectively configured, and the two is descending by concentration
It is successively slowly added into the sterile centrifugation tube of 15ml, while lymphocyte separation medium 5ml being taken to be added to the 15ml sterile centrifugation
In pipe.
(2) using sterile antibiotic physiological saline repeated flushing 8~10 times, then pig placenta is placed on superclean bench
Pig placental villi is cut using ophthalmology scissors and shreds into multistage.
(3) villus after shredding is immersed in 35~40 DEG C of incubator, includes that trypsase and DNA enzymatic are mixed in incubator
Liquid is closed, digestion time is 30~40min；
(4) it to DMEM/F12 culture solution termination digestion of the addition containing 10%FBS in cell suspension is obtained after digesting, blows and beats
At cell suspension；It after cell suspension is filtered by 100 mesh copper mesh, is transferred in centrifuge tube, 1000~1500r/min, centrifugation 3
~10min；
(5) supernatant is abandoned after being centrifuged, adds DMEM/F12 culture solution that cell is resuspended, cell suspension is respectively added slowly to
Percoll separating liquid and lymphocyte separation medium upper layer, continue to be centrifuged, 1500~2000r/min, are centrifuged 25~35min；
(6) the canescence cloud cellular layer and lymphocyte of two concentration liquid level intersections of Percoll separating liquid are drawn
Cellular layer between separating liquid and culture solution is washed twice, 1000~1500rpm with antibiotic salt water and culture solution, and centrifugation 5~
10min；
(7) into the cell suspension after washed, DMEM/F12 culture medium of the addition containing 20%FBS is resuspended, and adjustment cell is dense
Degree is inoculated in is coated with Collagen type-I (5-10U/cm2) and the 6 hole cell culture without collagen processing respectively to 1.0 × 106/ml
In plate, 2~3h is cultivated in 37 DEG C, 5%CO2 cell incubator.
In the method for building up of this boar source 3D placental organ model of the invention, the mixed liquor in step (3) is by containing
Example 1:1 is formed by volume for trypsin solution aqueous solution and aqueous solution containing DNA enzymatic, wherein aqueous solution containing trypsin solution
The concentration of middle trypsase is 0.25%, and the content of DNA enzymatic is 100U/ml in the aqueous solution containing DNA enzymatic.
The method for building up for implementing this boar source 3D placental organ model of the invention, has the advantages that
(1) cell line model and primary cell model single cell type, immortalization or cancerization feature, primary thin are overcome
Born of the same parents can not long-term cultivation and passage the shortcomings that, can permanently be passed on, be frozen with technical measure culture 3D pig source placenta organoid
Deposit, present invention firstly provides 3D pig placenta organoid will to basic research and it is applied research have important value.
(2) (syncytionboph-oblast and cytotrophoblast are thin for the nearly all functioning cell of placenta cells in occlusion body
Born of the same parents), the distinctive polypeptide of placenta and hormone can be secreted, hence it is evident that be better than existing 2D cell line and primary cell model.And the 3D
Organoid model cultivating system is by debugging, optimization, it is ensured that placenta organoid long-term cultivation stablizes passage, and can be used as and grind
Study carefully the model of pig placental function.
(3) the 3D pig placental organ culture system is derived from placenta stem-cell, and stem cell is divided into different placenta function
Energy cell includes plasmoditropho blast and cytotrophoblast, therefore is totally different from 2D cell model.
(4) 3D pig placental organ growth maintains 3D structure in Matrigel, therefore is closer to intracorporal feelings
Condition, 3D pig placenta organoid include plasmoditropho blast and cytotrophoblast, very close internal placenta structure.
(5) 3D pig placental organ culture system provided by the invention includes placenta stem-cell separation scheme and culture medium is
Completely new approach and formula.
Specific embodiment
According to summary of the invention, 3D pig placenta organoid is successfully turned out, it is seen that pig placenta organoid has apparent conjunction born of the same parents
Body trophoderm and cytotrophoblast functional areas.Specific implementation step are as follows: (1) separate placenta stem-cell, be wrapped in matrigel, add
Enter organoid growth factor, is put into cell incubator and cultivates；(2) it periodically takes pictures under an optical microscope；(3) copolymerization is burnt carries out
Immunofluorescence observes placenta organoid difference cell type.
The required 3D placental organ culture based component table of the present invention
Embodiment 1
The method for building up of one boar source 3D placental organ model, its step are as follows:
(1) the isotonic 30% and isotonic 60%Percoll of two parts of 3ml is respectively configured, and the two is descending by concentration
It is successively slowly added into the sterile centrifugation tube of 15ml, while lymphocyte separation medium 5ml being taken to be added to the 15ml sterile centrifugation
In pipe.
(2) pig placenta is placed on superclean bench using sterile antibiotic physiological saline repeated flushing 8 times, is then used
Ophthalmology scissors cut pig placental villi and shred into multistage.
(3) villus after shredding is immersed in 35 DEG C of incubator, includes trypsase and DNA enzymatic mixing in incubator
Liquid, digestion time 30min；
(4) it to DMEM/F12 culture solution termination digestion of the addition containing 10%FBS in cell suspension is obtained after digesting, blows and beats
It is transferred in centrifuge tube, 1000r/min after filtering cell suspension by 100 mesh copper mesh at cell suspension, is centrifuged 5min.
(5) supernatant is abandoned after being centrifuged, adds DMEM/F12 culture solution that cell is resuspended, cell suspension is respectively added slowly to
Percoll separating liquid and lymphocyte separation medium upper layer, continue to be centrifuged, 1500r/min, are centrifuged 25min.
(6) the canescence cloud cellular layer and lymphocyte of two concentration liquid level intersections of Percoll separating liquid are drawn
Cellular layer between separating liquid and culture solution is washed twice, 1000rpm with antibiotic salt water and culture solution, is centrifuged 5min.
(7) into the cell suspension after washed, DMEM/F12 culture medium of the addition containing 20%FBS is resuspended, and adjustment cell is dense
Degree is inoculated in is coated with Collagen type-I (5-10U/cm2) and the 6 hole cell culture without collagen processing respectively to 1.0 × 106/ml
In plate, 2h is cultivated in 37 DEG C, 5%CO2 cell incubator.
Embodiment 2
The method for building up of one boar source 3D placental organ model, its step are as follows:
(1) the isotonic 30% and isotonic 60%Percoll of two parts of 3ml is respectively configured, and the two is descending by concentration
It is successively slowly added into the sterile centrifugation tube of 15ml, while lymphocyte separation medium 5ml being taken to be added to the 15ml sterile centrifugation
In pipe.
(2) pig placenta is placed on superclean bench using sterile antibiotic physiological saline repeated flushing 10 times, is then made
Pig placental villi is cut with ophthalmology scissors and shreds into multistage.
(3) villus after shredding is immersed in 40 DEG C of incubator, includes trypsase and DNA enzymatic mixing in incubator
Liquid, digestion time 40min；
(4) it to DMEM/F12 culture solution termination digestion of the addition containing 10%FBS in cell suspension is obtained after digesting, blows and beats
It is transferred in centrifuge tube, 1500r/min after filtering cell suspension by 100 mesh copper mesh at cell suspension, is centrifuged 10min.
(5) supernatant is abandoned after being centrifuged, adds DMEM/F12 culture solution that cell is resuspended, cell suspension is respectively added slowly to
Percoll separating liquid and lymphocyte separation medium upper layer, continue to be centrifuged, 2000r/min, are centrifuged 35min.
(6) the canescence cloud cellular layer and lymphocyte of two concentration liquid level intersections of Percoll separating liquid are drawn
Cellular layer between separating liquid and culture solution is washed twice, 1000rpm with antibiotic salt water and culture solution, is centrifuged 5min.
(7) into the cell suspension after washed, DMEM/F12 culture medium of the addition containing 20%FBS is resuspended, and adjustment cell is dense
Degree is inoculated in is coated with Collagen type-I (5-10U/cm2) and the 6 hole cell culture without collagen processing respectively to 1.0 × 106/ml
In plate, 3h is cultivated in 37 DEG C, 5%CO2 cell incubator.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, made any modification, equivalent replacement and improvement etc. be should all be included in the protection scope of the present invention.
Claims (4)
1. the method for building up of a boar source 3D placental organ model, which is characterized in that its step are as follows:
(1) the isotonic 30% and isotonic 60%Percoll of two parts of 3ml is respectively configured, and the two is descending successively by concentration
It is slowly added into the sterile centrifugation tube of 15ml, while lymphocyte separation medium 5ml being taken to be added in the 15ml sterile centrifugation tube；
(2) using sterile antibiotic physiological saline repeated flushing 8~10 times, then pig placenta tissue is placed on superclean bench
Pig placental villi is cut using ophthalmology scissors and shreds into multistage；
(3) villus after shredding is immersed in 35~40 DEG C of incubator, includes trypsase and DNA enzymatic mixing in incubator
Liquid, digestion time are 30~40min；
(4) it to DMEM/F12 culture solution termination digestion of the addition containing 10%FBS in cell suspension is obtained after digesting, blows and beats at thin
Born of the same parents' suspension；After cell suspension is filtered by 100 mesh copper mesh, it is transferred in centrifuge tube, 1000~1500r/min, centrifugation 3~
10min；
(5) supernatant is abandoned after being centrifuged, adds DMEM/F12 culture solution that cell is resuspended, cell suspension is respectively added slowly to Percoll points
Chaotropic and lymphocyte separation medium upper layer, 1500~2000r/min are centrifuged 25~35min；
(6) the canescence cloud cellular layer and separation of lymphocytes of two concentration liquid level intersections of Percoll separating liquid are drawn
Cellular layer between liquid and culture solution is washed twice, 1000~1500rpm with antibiotic salt water and culture solution, and centrifugation 5~
10min；
(7) into the cell suspension after washed, DMEM/F12 culture medium of the addition containing 20%FBS is resuspended, and adjustment cell concentration is extremely
1.0 × 106/ml is inoculated in is coated in Collagen type-I (5-10U/cm2) and 6 porocyte culture plates handled without collagen respectively,
2~3h is cultivated in 37 DEG C, 5%CO2 cell incubator.
2. method for building up according to claim 1, which is characterized in that be pig Placenta samples in step (2).
3. method for building up according to claim 1, which is characterized in that the mixed liquor in step (3) is by molten containing trypsase
Example 1:1 is formed by volume for liquid aqueous solution and aqueous solution containing DNA enzymatic, wherein trypsase in aqueous solution containing trypsin solution
Concentration be 0.25%, the content of DNA enzymatic is 100U/ml in the aqueous solution containing DNA enzymatic.
4. method for building up according to claim 1, which is characterized in that the 3D pig placenta organoid provided in step (1-7)
Cultivating system includes placenta stem-cell separation scheme and culture medium prescription.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110669124A (en) * | 2019-10-17 | 2020-01-10 | 同济大学 | New application of gene TBX3 |
| CN111534489A (en) * | 2020-04-29 | 2020-08-14 | 清华大学 | T lymphocyte amplification method based on 3D printing |
| CN112538457A (en) * | 2020-12-23 | 2021-03-23 | 中国科学院亚热带农业生态研究所 | Pig liver and spleen duct stem cell separation and three-dimensional organoid culture method |
| CN112608885A (en) * | 2020-12-23 | 2021-04-06 | 中国科学院亚热带农业生态研究所 | Porcine fundus stomach gland stem cell separation and three-dimensional organoid culture method |
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| CN105087470A (en) * | 2015-07-31 | 2015-11-25 | 何静 | Method for separating and purifying human embryo trophoblast and placental mesenchymal stem cells |
| CN107236704A (en) * | 2017-06-15 | 2017-10-10 | 博雅干细胞科技有限公司 | From the method for placenta separating mesenchymal stem cell and the digestive enzyme compositions used |
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| CN105087470A (en) * | 2015-07-31 | 2015-11-25 | 何静 | Method for separating and purifying human embryo trophoblast and placental mesenchymal stem cells |
| CN107236704A (en) * | 2017-06-15 | 2017-10-10 | 博雅干细胞科技有限公司 | From the method for placenta separating mesenchymal stem cell and the digestive enzyme compositions used |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110669124A (en) * | 2019-10-17 | 2020-01-10 | 同济大学 | New application of gene TBX3 |
| CN111534489A (en) * | 2020-04-29 | 2020-08-14 | 清华大学 | T lymphocyte amplification method based on 3D printing |
| CN112538457A (en) * | 2020-12-23 | 2021-03-23 | 中国科学院亚热带农业生态研究所 | Pig liver and spleen duct stem cell separation and three-dimensional organoid culture method |
| CN112608885A (en) * | 2020-12-23 | 2021-04-06 | 中国科学院亚热带农业生态研究所 | Porcine fundus stomach gland stem cell separation and three-dimensional organoid culture method |
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Application publication date: 20190816 |