CN105861426A - Isolated culturing method of oral mucosa epithelium stem cells - Google Patents
Isolated culturing method of oral mucosa epithelium stem cells Download PDFInfo
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- CN105861426A CN105861426A CN201610338840.8A CN201610338840A CN105861426A CN 105861426 A CN105861426 A CN 105861426A CN 201610338840 A CN201610338840 A CN 201610338840A CN 105861426 A CN105861426 A CN 105861426A
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Abstract
The invention relates to the technical field of cell culturing, in particular to an isolated culturing method of oral mucosa epithelium stem cells. The isolated culturing method includes the steps that an oral mucosa epithelium tissue is digested through trypsin and then digested through neutral protease, cell culturing is carried out after digestion is stopped, and the oral mucosa epithelium stem cells are obtained. By means of the isolated culturing method, damage, caused when the tissue is severely digested through enzymes, to cells can be effectively relieved, and therefore the more oral mucosa epithelium stem cells high in activity can be obtained.
Description
Technical field
The present invention relates to technical field of cell culture, dividing particularly to a kind of Oral mucosa keratinocyte stem cell
From cultural method.
Background technology
The numerous disease of Oral and Maxillofacial Surgery the most usually leaves over the lower oral cavity wound surface needing and repairing.
Although grafts is wear-resisting, but Post operation often shrinks more serious, and even at intraoral environment,
It also there will be the phenomenon of hair growth, can't metaplasia be oral mucosa completely.Additionally, it is all with utilizing
The oral mucosa enclosed is transplanted and is compared, and graft area is also transplanted not as oral mucosa in the compatibility and color
Effective.The Shi Gong district more allowing patient be difficult to accept often has some not accommodate the generation of complication.Mucosa
Transplanting is in its natural environment, is therefore more particularly suitable, but the available tissue in oral cavity self
Amount is very limited amount of, and need to open up the second art district.In recent years, the research of tissue stem cell has become as
Domestic scholars focus of attention, the successful cultivation of Oral mucosa keratinocyte stem cell is likely brand-new for building
Artificial mucous membrane tissue brings hope, thus solves an above difficult problem.
At present, the isolated culture method of conventional Oral mucosa keratinocyte stem cell is: take part normal person's cheek
Portion's mucosa specimen, is placed in mucous membrane tissue in the pancreatin of 0.25%, and overnight, Hanks liquid is light in 4 DEG C of digestion
Wash 3 times, de-epithelization, blow and beat into single cell suspension with dropper, be filtered to remove bulk tissue and fragment,
Suck cell suspension Auto-counting of Cells instrument and carry out the calculating of cell quantity and vigor, be then inoculated in and mould
Material culture bottle, condition of culture is IMDM+10% hyclone, 37 DEG C, 5%CO2.After cultivating 6h,
Discard suspension cell gently, continue to cultivate by attached cell, after major part at the bottom of covering with bottle, trypsinization,
Routine passage.Within 3 days one, change liquid.
Zhang Lan is in " preliminary study of rabbit Oral mucosa keratinocyte stem cell cultivation structure organizational project mucosa "
Report the isolated culture method of a kind of rabbit Oral mucosa keratinocyte stem cell, particularly as follows:
1) acquisition of rabbit Oral mucosa keratinocyte
Taking healthy rabbits, male and female do not limit, body weight 0.5～1.0kg, and auricular vein injection air is put to death.Oral cavity
Drawing materials of mucosal epithelial cells: 0.5% iodophor disinfection oral cavity, sterile working takes out rabbit bilateral cheek mucosa and is placed in
Normal saline is sent to clean room.With the PBS liquid containing gentamycin 2000U/mL on superclean bench
Soak oral mucosa 10 minutes, then float with the PBS containing penicillin, streptomycin (each 100U/mL)
Wash 3 times.Remove sub-mucosal tissues with eye scissors, then by group under stereomicroscope after PBS flushing as far as possible
Knit block to be placed in the aseptic bottle containing 0.25% neutral protease Dispase.4 DEG C of refrigerators digest 16～18 little
Shi Hou, with ophthalmic tweezers separation epidermis under stereomicroscope, i.e. can obtain the purer mouth comprising basal layer
Chamber mucosal epithelium.
2) rabbit Buccal mucosa cell separates, cultivates
Oral mucosa epidermal tissue under separating is divided into equal two parts, shreds with eye scissors, uses 0.25% pancreas
Protease-0.02%EDTA mixed in equal amounts Digestive system digests.37 DEG C digestion 5～8 minutes after add
DMEM (containing 10%FBS) terminates trypsinization.1000rpm is centrifuged 5 minutes, abandons supernatant, PBS
Washing.First group of culture fluid is epithelial cell culture fluid, and second group of culture fluid is Defined K-SFM.Two
Group cell is separately added into different culture fluid, the tissue of piping and druming digestion and cell so that it is dispersion is free, resuspended
Cell is in culture fluid.Blood cell plate calculates cell number, and platform expects blue living cells dyeing, then with 105Individual/mL
Cell number be inoculated into culture dish.Within second day, change liquid and remove non-attached cell, the next day of later, change liquid.
But, the stem cell population separated in the isolated culture method of above-mentioned Oral mucosa keratinocyte stem cell is few,
Vigor is the highest.Therefore, how to obtain greater number and the high Oral mucosa keratinocyte stem cell of vigor becomes and grinds
The focus studied carefully.
Summary of the invention
In view of this, the invention provides the isolated culture method of a kind of Oral mucosa keratinocyte stem cell.Should
Isolated culture method can obtain greater number and the high Oral mucosa keratinocyte stem cell of vigor.
In order to realize foregoing invention purpose, the present invention provides techniques below scheme:
The invention provides the isolated culture method of a kind of Oral mucosa keratinocyte stem cell, comprise the steps:
After Oral mucosa keratinocyte tissue is used trypsinization, then use neutral protein enzymic digestion, terminate disappearing
Cell cultivation is carried out, it is thus achieved that Oral mucosa keratinocyte stem cell after change.
As preferably, the temperature of trypsinization is 4 DEG C, and the time is 12～18h.
Preferably, the time of trypsinization is 12h.
The present invention provide some embodiments in, pancreatin be pancreatin weight/mass percentage composition be the pancreatin of 0.25%
-EDTA。
As preferably, the temperature of neutral protein enzymic digestion is 37 DEG C, and concussion rotating speed is 200～500rpm, time
Between be 15～30min.
Preferably, the concussion rotating speed of neutral protein enzymic digestion is 200rpm, and the time is 15min.
In some embodiments that the present invention provides, neutral protease is neutral protease weight/mass percentage composition
It it is the Digestive system of 0.25%.
As preferably, it is serum-free medium that cell cultivates the culture medium used.
As preferably, the formula of serum-free medium is Lonza culture medium.
As preferably, terminate digestion and use the complete medium containing serum.
As preferably, the inoculum density that cell is cultivated is (1～10) × 103Individual/cm2。
Preferably, the inoculum density that cell is cultivated is 1 × 104Individual/cm2。
In some embodiments that the present invention provides, cell is cultivated and is: after cell is inoculated in culture medium,
At 37 DEG C, 5%CO2Under the conditions of carry out cell cultivation, within second day, change liquid, remove attached cell, later every
Day changes liquid, co-cultures 7～14 days.
The invention provides the isolated culture method of a kind of Oral mucosa keratinocyte stem cell.This separation and Culture side
Method includes: after Oral mucosa keratinocyte tissue is used trypsinization, then use neutral protein enzymic digestion, eventually
Only carry out cell cultivation after digestion, it is thus achieved that Oral mucosa keratinocyte stem cell.The invention have the benefit that
1, in the isolated culture method that the present invention provides, digestion method uses first with in trypsinization, then employing
Property protease digests, and can effectively alleviate enzyme acutely digests the tissue damage to cell, thus can obtain
Greater number and the high Oral mucosa keratinocyte stem cell of vigor；
2, culture medium used in the present invention is serum-free medium, is prevented effectively from what cell was brought by serum
External source is polluted.
Accompanying drawing explanation
Fig. 1 shows the cell primary growthform that test group separates；
Fig. 2 shows the viability examination result of Oral mucosa keratinocyte stem cell；
Fig. 3 shows the streaming qualification result of Oral mucosa keratinocyte stem cell；Wherein Fig. 3-1,3-2,3-3 are comparison
Group streaming qualification result, 3-1 shows the expression that blank, 3-2 show CD90, CD45,3-3 show CD73,
The expression of HLA-DR；Fig. 3-4,3-5,3-6 are sample sets streaming qualification result, and 3-4 shows blank right
According to, 3-5 shows the expression of CD90, CD45, and 3-6 shows the expression of CD73, HLA-DR；
Fig. 4 shows the one-tenth fat differentiation qualification result of Oral mucosa keratinocyte stem cell；
Fig. 5 shows the Osteoblast Differentiation qualification result of Oral mucosa keratinocyte stem cell.
Detailed description of the invention
The invention discloses the isolated culture method of a kind of Oral mucosa keratinocyte stem cell, people in the art
Member can use for reference present disclosure, is suitably modified technological parameter and realizes.Special needs to be pointed out is, all classes
As replace and change apparent to those skilled in the art, they are considered as being included in
The present invention.Method and the application of the present invention are described by preferred embodiment, and related personnel is bright
Show off one's talent or competence and in without departing from present invention, spirit and scope, method described herein and application are modified
Or suitably change and combination, realize and apply the technology of the present invention.
Term is explained:
It is strong that mescenchymal stem cell is that a class has self renewal, multiplication capacity, and Multidirectional Differentiation ability is dry
Cell, is widely present in whole body connective tissue and organ interstitial.In oral mucosa epithelial cell, deposit
At a small amount of mescenchymal stem cell, there is the surface marker that mescenchymal stem cell is common, and exist multidirectional
The ability of differentiation；
Neutral protease (Dispase), also referred to as Bacillus polymyxa Neutral proteinase, is a kind of nonspecific metalloproteases,
It is used to peptic cell epimatrix from various different tissues or organ, discharges and prepare primary unicellular；
Or it is used for the cell harvesting during cell is cultivated or inoculation transfer；When also being used for preventing suspension cell culture
Cell accident conglomeration.
Biomaterial used and examination in the isolated culture method of the Oral mucosa keratinocyte stem cell that the present invention provides
Agent all can be buied by market.
Below in conjunction with embodiment, the present invention it is expanded on further:
Embodiment 1
(1) separation and Culture Oral mucosa keratinocyte stem cell
Test and be divided into test group, matched group 1, matched group 2:
1, the method for the separation and Culture Oral mucosa keratinocyte stem cell of test group is:
Take normal person buccal mucous membrane tissue, be placed in containing penicillin, streptomycin (each 100U/mL) physiology salt
In water, low-temperature transport to laboratory, in aseptic operating platform, by mucous membrane tissue transfer put containing penicillin,
The PBS of streptomycin (each 100U/mL) cleans 2-3 time；Mucous membrane tissue is placed on 0.25% pancreas
In enzyme-EDTA, after being placed in 4 DEG C of refrigerators digestion 12 hours, remove table with ophthalmic tweezers under the microscope
Skin, obtains Oral mucosa keratinocyte tissue.Again with containing 0.25% neutral protease Dispase at 37 DEG C, 200rpm
Isothermal vibration digestion 15min, terminates neutral protease by Serum-free complete medium (containing serum substitute)
Digestion.1000rpm is centrifuged 5 minutes, abandons supernatant, PBS washing.Add epithelial cell culture fluid resuspended carefully
Born of the same parents, and count blood cell plate calculating cell number, platform is expected that orchid carries out living cells dyeing, is calculated cell viability and number
Amount, then with 104Individual/cm2Cell number be inoculated into culture dish.Within second day, change liquid and remove non-attached cell,
Liquid is changed the next day of later.About 7-14d under the microscope it is observed that clone formation, when cell grows to
When 80%-90% converges, 0.125% trypsin digestion and cell, carry out Secondary Culture.Fig. 1 is cell primary
Growthform, form meets the feature of mescenchymal stem cell.
2, the method for the separation and Culture Oral mucosa keratinocyte stem cell of matched group 1 is:
Take part normal person buccal mucosa specimen, mucous membrane tissue is placed in the pancreatin of 0.25%, 4 DEG C of digestion
Overnight, Hanks liquid rinses 3 times, and de-epithelization is blown and beaten into single cell suspension with dropper, is filtered to remove
Bulk tissue and fragment, suck cell suspension Auto-counting of Cells instrument and carry out the meter of cell quantity and vigor
Calculating, be then inoculated in plastic culture bottle, inoculum density is 104Individual/cm2, condition of culture is IMDM+10%
Hyclone, 37 DEG C, 5%CO2.After cultivating 6h, discard suspension cell gently, attached cell is continued
Continuous cultivate, after major part at the bottom of covering with bottle, trypsinization, routine passage.Within 3 days one, change liquid.Primary carefully
The form of born of the same parents is close with Fig. 1, meets the feature of mescenchymal stem cell.
3, the method for the separation and Culture Oral mucosa keratinocyte stem cell of matched group 2 is:
1) acquisition of Oral mucosa keratinocyte
Take part normal person buccal mucosa specimen, steep by the PBS immersion containing gentamycin 2000U/mL
Oral mucosa 10 minutes, then rinse 3 times with the PBS containing penicillin, streptomycin (each 100U/mL).
Remove sub-mucosal tissues with eye scissors under stereomicroscope after PBS flushing as far as possible, then piece of tissue is placed in
In aseptic bottle containing 0.25% neutral protease Dispase.After 4 DEG C of refrigerators digest 16 hours, stereoscopic
With ophthalmic tweezers separation epidermis under microscope, i.e. can obtain comprising the purer Oral mucosa keratinocyte group of basal layer
Knit.
2) Buccal mucosa cell separates, cultivates
Oral mucosa epidermal tissue under separating is divided into equal two parts, shreds with eye scissors, uses 0.25% pancreas
Protease-0.02%EDTA mixed in equal amounts Digestive system digests.37 DEG C digestion 8 minutes after add DMEM
(containing 10%FBS) terminates trypsinization.1000rpm is centrifuged 5 minutes, abandons supernatant, PBS washing.
First group of culture fluid is epithelial cell culture fluid, and second group of culture fluid is Defined K-SFM.Two groups of cells
Be separately added into different culture fluid, the tissue of piping and druming digestion and cell so that it is dispersion is free, re-suspended cell in
In culture fluid.Blood cell plate calculates cell number, and platform expects blue living cells dyeing, then with 104Individual/cm2Cell
Number is inoculated into culture dish.Within second day, change liquid and remove non-attached cell, the next day of later, change liquid.Primary cell
Form is close with Fig. 1, meets the feature of mescenchymal stem cell.
(2) cell viability detection test
The cell trypan blue of primary separation in test group, matched group 1, matched group 2 is carried out cell viability
Detection, result is shown in Fig. 2.
As in figure 2 it is shown, the primary cell vigor that test group separates is higher than in matched group 1 and matched group 2
Cell viability.
(3) Oral mucosa keratinocyte stem cell P1 identifies for the streaming of cell
When test group P1 grows to 80%～90% for cell, with collecting cell after 0.25% trypsinization,
Take two 1.5mL EP pipes, often pipe 2 × 105Individual cell, with dye solution (10% hyclone
+ 90%PBS) wash twice, often pipe adds 200 μ L dye solution, and sample is separately added into following four antibody
The each 2 μ L of CD90, CD73, HLA-DR, CD45, negative control is not added with antibody, hatches 20min for 4 DEG C,
Wash twice with dye solution, the most resuspended by 500 μ L DMEM culture medium, gather with flow cytometer
100000 cells, detection CD90, CD73, HLA-DR, CD45 etc. are in pork fat stem cell
Expression.Result is shown in Fig. 3.
From the result of Fig. 3 it can be seen that sample high expressed CD73, CD90；Low expression of HLA-DR, CD45,
Meeting oral epithelium stem cell properties, the present invention can obtain the Buccal mucosa cell that purity is higher.
(4) fat differentiation is become to identify
Collect the epithelial stem cell in test group P3 generation, with 1 × 103/cm2Cell density be inoculated in 24 orifice plates,
When cell confluency degree reaches 100%, use adipogenic induction culture fluid instead and cultivate, within every 2-3 days, change fresh induction
Culture medium, uses oil red O stain to detect into fat differentiated result after 2 weeks.Fig. 4 is cell shape after adipogenic induction
State change (× 200).
From fig. 4, it can be seen that in vitro in adipose cell Induction Process, cellular morphology, from becoming threadiness, contracts
Short becoming short fusiformis, after induction 15d, visible most cells is full of red oil droplet cavity.
(5) Osteoblast Differentiation is identified
Collect the epithelial stem cell in test group P3 generation, respectively with 1 × 103/cm2Cell density be inoculated in 24
In orifice plate, when cultivating to 60～70% fusion, use osteogenic induction culture fluid instead, within every 3 days, change fresh one-tenth
Self-bone grafting culture medium, uses Alizarin red staining detection Osteoblast Differentiation result after 3 weeks.Result is shown in Fig. 5.
As seen from Figure 5, during Oral mucosa keratinocyte stem cell Osteoinductive differentiation in vitro, 21 days
The obvious Mineral nodules of Shi Kejian, the calcium tuberosity that can be formed with Alizarin red staining dyes redness.
The above is only the preferred embodiment of the present invention, it is noted that general for the art
For logical technical staff, under the premise without departing from the principles of the invention, it is also possible to make some improvement and profit
Decorations, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (10)
1. the isolated culture method of an Oral mucosa keratinocyte stem cell, it is characterised in that include walking as follows
Rapid:
After Oral mucosa keratinocyte tissue is used trypsinization, then use neutral protein enzymic digestion, terminate disappearing
Cell cultivation is carried out, it is thus achieved that Oral mucosa keratinocyte stem cell after change.
Isolated culture method the most according to claim 1, it is characterised in that described trypsinization
Temperature is 4 DEG C, and the time is 12～18h.
Isolated culture method the most according to claim 1, it is characterised in that described pancreatin is pancreatin
Weight/mass percentage composition is the pancreas enzyme-EDTA of 0.25%.
Isolated culture method the most according to claim 1, it is characterised in that described neutral protease
The temperature of digestion is 37 DEG C, and concussion rotating speed is 200～500rpm, and the time is 15～30min.
Isolated culture method the most according to claim 1, it is characterised in that described neutral protease
It is the Digestive system of 0.25% for neutral protease weight/mass percentage composition.
Isolated culture method the most according to claim 1, it is characterised in that described cell is cultivated and adopted
Culture medium be serum-free medium.
Isolated culture method the most according to claim 6, it is characterised in that described serum-free culture
The formula of base is Lonza culture medium.
Isolated culture method the most according to claim 1, it is characterised in that described termination digestion is adopted
With the complete medium containing serum.
Isolated culture method the most according to claim 1, it is characterised in that described cell is cultivated
Inoculum density is (1～10) × 104Individual/cm2。
Isolated culture method the most according to claim 1, it is characterised in that described cell is cultivated
For: after cell is inoculated in culture medium, at 37 DEG C, 5%CO2Under the conditions of carry out cell cultivation, second day
Change liquid, remove attached cell, change liquid next day of later, co-culture 7～14 days.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107384853A (en) * | 2017-08-08 | 2017-11-24 | 安徽惠恩生物科技股份有限公司 | A kind of Buccal mucosa cell reprogramming is the method for multipotential stem cell |
| CN108498863A (en) * | 2017-07-11 | 2018-09-07 | 上海白衣缘生物工程有限公司 | A kind of oral cavity sticking patch preparation method of compound oral mucosa epithelial cell |
| CN113025558A (en) * | 2021-02-23 | 2021-06-25 | 王姗 | Auxiliary reagent for detecting desquamation cells of precancerous lesions of oral mucosa |
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| CN104862269A (en) * | 2015-06-12 | 2015-08-26 | 李超 | Method for culturing oral mucosa cells |
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2016
- 2016-05-20 CN CN201610338840.8A patent/CN105861426A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104862269A (en) * | 2015-06-12 | 2015-08-26 | 李超 | Method for culturing oral mucosa cells |
Non-Patent Citations (2)
| Title |
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| 张岚 等: "兔口腔黏膜上皮细胞的体外培养方法", 《山西医科大学学报》 * |
| 陈文 等: "人体口腔粘膜上皮细胞体外培养和鉴定", 《现代口腔医学杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108498863A (en) * | 2017-07-11 | 2018-09-07 | 上海白衣缘生物工程有限公司 | A kind of oral cavity sticking patch preparation method of compound oral mucosa epithelial cell |
| CN107384853A (en) * | 2017-08-08 | 2017-11-24 | 安徽惠恩生物科技股份有限公司 | A kind of Buccal mucosa cell reprogramming is the method for multipotential stem cell |
| CN113025558A (en) * | 2021-02-23 | 2021-06-25 | 王姗 | Auxiliary reagent for detecting desquamation cells of precancerous lesions of oral mucosa |
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Application publication date: 20160817 |