CN107841487A - The method for cultivating taste stem cells - Google Patents
The method for cultivating taste stem cells Download PDFInfo
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- CN107841487A CN107841487A CN201711334289.0A CN201711334289A CN107841487A CN 107841487 A CN107841487 A CN 107841487A CN 201711334289 A CN201711334289 A CN 201711334289A CN 107841487 A CN107841487 A CN 107841487A
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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
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Abstract
A kind of method for cultivating taste stem cells, pass through the taste stem cells of tongue ring-type nipple taste bud bottom after in-vitro separation, blown and beaten and filter out individual cells, and cultivate in the culture medium containing matrigel, after 12~14 days, taste stem cells are bred as the organoid containing ripe gustatory through differentiation.A kind of active stimulate two kinds of sweet stimulus and bitter taste of taste stem cells through the inventive method culture has reaction.
Description
Technical field
The invention belongs to cell biology, and in particular to a kind of method for cultivating taste stem cells.
Background technology
The generation of the sense of taste is to be discharged by gustatory and transmitted Taste Signals to cause.The damage of gustatory and missing cause
Taste dysfunction even lacks.Gustatory continues ceaselessly renewal and differentiation, and the update cycle is 14 days.Animal passes through tongue
On taste bud experience various tastes, including hardship, sweet tea, salty, sour, fresh etc..Taste bud be mainly distributed on before tongue fungiform papilla and after
The filiform papillae and ring-type nipple of tongue.Taste buds cell is divided into four major classes by its form and function.The first kind is sertoli cell, mark
Albumen is NTPDaseII；Second class is taste receptor cells, and mainly bitter taste, sweet taste and delicate flavour are stimulated and made a response, marks egg
White is Gustducin, T1r3, P1c β 2 and Trpm5；3rd class is presynaptic cell, and mainly saline taste and tart flavour are stimulated and made
Reaction, labelled protein is Ca4 and 5-HT.A kind of cell positioned at taste bud bottom is basal cell, or taste stem cells, can be with
Persistently break up gustatory, taste bud is experienced the stimulation of various tastes.
The content of the invention
It is an object of the invention to provide a kind of method for cultivating taste stem cells, has the functional maturation sense of taste to obtain
Cell.
The present invention by the taste stem cells of tongue ring-type nipple taste bud bottom after in-vitro separation, blown and beaten filter out it is single
Cell, and cultivate in the culture medium containing matrigel, after 12~14 days, taste stem cells through differentiation, breed for containing into
The organoid of ripe gustatory.
Culture base system containing matrigel culture medium based on DMEM/F12, is additionally added various ingredients to stimulate the sense of taste to do
The propagation of cell and differentiation.Matrigel carries out dimensional culture for taste stem cells and provides nutrition, also acts as fixed sertoli cell shape
The effect of state.The each component and its content added in basal medium is as follows：
Brief description of the drawings
Fig. 1 is the cell picture of taste stem cells culture different number of days, reference numerals 1,2,3,4,7,9 and 15 wherein in figure
The cell image of culture tracking shooting in the 1st day, the 2nd day, the 3rd day, the 4th day, the 7th day, the 9th day and the 15th day is represented respectively；
Fig. 2 is the taste stem cells immunostaining image of culture 10 days, is schemed in A, " BrdU ", " Sox2 " and " DAPI " difference
Represent the method that mark uses, i.e. " BrdU " mark proliferative cell, " Sox2 " labeled stem cells, and " DAPI " mark cell
Core；Scheme in B, " K8 ", " K5 " and " DAPI " represents the method that mark uses, i.e. " K8 " mark differentiation gustatory, " K5 " respectively
Marking substrates cell, " DAPI " mark nucleus；
Fig. 3 is the taste stem cells immunostaining picture of culture 14 days, wherein " Gustducin ", " Car4 " and " K8 " point
The method used, i.e. " Gustducin " mark II type gustatories Biao Shi not be marked, " Car4 " marks type III gustatory,
" K8 " mark differentiation gustatory；
Fig. 4 is the culture taste stem cells calcium imaging results of 14 days, is schemed in A, " Ace.K (Acesulfame K) " and
" Sucralose " represents sweet taste reagent, and noble cells has reaction to two kinds of sweet stimulus；Scheme in B, " Den.
(Denatonium) " it is bitter taste reagent, noble cells, which is stimulated bitter taste, reaction, and the higher reaction of concentration is bigger；U73122 is
The pathway inhibitors of Plc β 2, bitter taste caused by Den. can be suppressed and reacted, reacting recovery after U73122 is removed, show what is differentiated
Gustatory is active.
Embodiment
Technical scheme is described in detail below in conjunction with accompanying drawing.The embodiment of the present invention only to illustrate the present invention skill
Art scheme and it is unrestricted, although the present invention is described in detail with reference to preferred embodiment, one of ordinary skill in the art
It should be appreciated that the technical scheme of invention can be modified or equivalent substitution, without departing from the essence of technical solution of the present invention
God and scope, it all should cover in scope of the presently claimed invention.
1) mouse anesthesia is lethal, scissors cuts off the cheek of mouse two, cuts tongue from root of the tongue portion, is placed in the PBS of precooling on ice
In.Washed twice, be placed in Tyrode ' s buffer solutions with ice-cold PBS.
2) with 1ml syringes to lower injection enzyme liquid among tongue epidermis and muscle layer, 37 DEG C are placed 15 minutes.
3) rear tongue ring-type mamillary region is cut with microscissors, and is shredded into as far as possible small tissue block.
4) 37 DEG C of 0.25% pancreatin digestion tissue 15 minutes.
5) the DMEM culture mediums containing FBS are added and terminates digestion.
6) 1200rpm is centrifuged 5 minutes, precipitates tissue.
7) pancreatin is removed, adds 1ml complete mediums, is blown and beaten 20 times with glass pipette, obtains cell suspension.
8) cell suspension is obtained into single cell suspension respectively by 70 μm and 40 μm of cell screen clothes.
9) matrigel is added in single cell suspension, cell is inoculated in low absorption orifice plate (such as：24 holes of low adsorption capacity
Plate) on, it is placed in 37 DEG C of incubators and cultivates.
10) cell is carried out every three days changing liquid, started to grow gustatory, the 14th day gustatory to the 8th day or so
It is ripe and active.
Above-mentioned steps 1) described in Tyrode ' s buffer solutions (pH7.4) formula be：145mM NaCl、5mM KCl、10mM
Hepes buffer solutions, 5mM NaHCO3, 10mM acetonates and 10mM glucose.
Above-mentioned steps 2) in enzyme liquid be：Contain separation enzyme (2mg/mL, Roche) and clostridiopetidase A (1mg/mL, Roche)
Tyrode ' s buffer solutions.
Above-mentioned steps 9), matrigel accounts for the 5w/w% of culture volume.
As shown in Figure 1 to 4, empirical tests, the gustatory differentiated is active, and lactation is moved for gustatory after culture
Thing gustatory has the ability of continuous updating.The present embodiment obtains the taste stem cells of mouse by in-vitro separation, has body
Outer propagation and differentiation capability, and can be divided into active gustatory in the case where no nerve is supported.This side
Method simulates internal gustatory generation and the process of development, elaborates the atomization from taste stem cells to gustatory,
Obtain gustatory in vitro with the method, can be impaired to ageusia, the sense of taste etc. sense of taste relevant disease provide Research Thinking with
Foundation.
Claims (8)
- A kind of 1. method for cultivating taste stem cells, it is characterised in that pass through the taste of tongue ring-type nipple taste bud bottom after in-vitro separation Feel stem cell, blown and beaten and filter out individual cells, and cultivate in the culture medium containing matrigel, after 12~14 days, taste Feel that stem cell through differentiation, breeds as the organoid containing ripe gustatory.
- 2. the method for culture taste stem cells according to claim 1, it is characterised in that described culture medium is with DMEM/ Culture medium based on F12.
- 3. the method for culture taste stem cells according to claim 1, it is characterised in that described culture medium adds growth The factor, apoptosis inhibitor, R-spondin and Noggin.
- 4. the method for culture taste stem cells according to claim 1, it is characterised in that described culture medium is with DMEM/ Culture medium based on F12, it is additionally added growth factor, apoptosis inhibitor, R-spondin and Noggin.
- 5. the method for culture taste stem cells according to claim 4, it is characterised in that described apoptosis inhibitor For Y27632.
- 6. the method for culture taste stem cells according to claim 4, it is characterised in that described growth factor is selected from The one or more of N2, B27, Jagged-1 albumen, N-acetylcystein and EGF.
- 7. the method for culture taste stem cells according to claim 1, it is characterised in that be additionally added following component：
- 8. the method for culture taste stem cells according to claim 1, it is characterised in that described culture medium is with DMEM/ Culture medium based on F12, it is additionally added following component：
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| CN201711334289.0A CN107841487A (en) | 2017-12-13 | 2017-12-13 | The method for cultivating taste stem cells |
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| CN201711334289.0A CN107841487A (en) | 2017-12-13 | 2017-12-13 | The method for cultivating taste stem cells |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109679913A (en) * | 2019-02-26 | 2019-04-26 | 复旦大学附属眼耳鼻喉科医院 | Smell stem cell three-dimensional culture method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101460635A (en) * | 2006-06-08 | 2009-06-17 | 塞诺米克斯公司 | Rationale, methods, and assays for identifying novel taste cell genes and salty taste receptor targets and assays using these identified genes or gene products |
| CN102439135A (en) * | 2009-02-03 | 2012-05-02 | 荷兰皇家科学院 | Culture medium for epithelial stem cells and organoids comprising said stem cells |
| CN103695369A (en) * | 2013-12-31 | 2014-04-02 | 章毅 | Umbilical cord mesenchymal stem cell in-vitro culture and amplification method |
| WO2015173425A1 (en) * | 2014-05-16 | 2015-11-19 | Koninklijke Nederlandse Akademie Van Wetenschappen | Improved culture method for organoids |
-
2017
- 2017-12-13 CN CN201711334289.0A patent/CN107841487A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101460635A (en) * | 2006-06-08 | 2009-06-17 | 塞诺米克斯公司 | Rationale, methods, and assays for identifying novel taste cell genes and salty taste receptor targets and assays using these identified genes or gene products |
| CN102439135A (en) * | 2009-02-03 | 2012-05-02 | 荷兰皇家科学院 | Culture medium for epithelial stem cells and organoids comprising said stem cells |
| CN103695369A (en) * | 2013-12-31 | 2014-04-02 | 章毅 | Umbilical cord mesenchymal stem cell in-vitro culture and amplification method |
| WO2015173425A1 (en) * | 2014-05-16 | 2015-11-19 | Koninklijke Nederlandse Akademie Van Wetenschappen | Improved culture method for organoids |
Non-Patent Citations (4)
| Title |
|---|
| EITARO AIHARA ET AL.: "Characterization of stem/progenitor cell cycle using murine circumvallate papilla taste bud organoid", 《SCIENTIFIC REPORTS》 * |
| HAKAN OZDENER ET AL.: "Characterization and Long-Term Maintenance of Rat Taste Cells in Culture", 《CHEM SENSES》 * |
| 廖贵清等: "大鼠舌背上皮细胞体外培养及其生物学特性观察", 《中国口腔颌面外科杂志》 * |
| 秦玉梅等: "小鼠味蕾细胞分离及体外培养方法", 《细胞生物学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109679913A (en) * | 2019-02-26 | 2019-04-26 | 复旦大学附属眼耳鼻喉科医院 | Smell stem cell three-dimensional culture method |
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