CN108277202A - A kind of cultural method of dental pulp mescenchymal stem cell - Google Patents
A kind of cultural method of dental pulp mescenchymal stem cell Download PDFInfo
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- CN108277202A CN108277202A CN201810051970.2A CN201810051970A CN108277202A CN 108277202 A CN108277202 A CN 108277202A CN 201810051970 A CN201810051970 A CN 201810051970A CN 108277202 A CN108277202 A CN 108277202A
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Abstract
A kind of cultural method of dental pulp mescenchymal stem cell, includes the following steps：Prepare dental pulp sample, be separately cultured dental pulp mescenchymal stem cell, colonized culture detaches dental pulp mescenchymal stem cell, the detection of dental pulp mescenchymal stem cell, the detection dyeing of dental pulp mescenchymal stem cell.The beneficial effects of the present invention are：1, close using being arranged between the cell of clone and separate culture, central part cell boundary is unclear.Rounded or irregular and formation cellular nodules sample, peripheral portion cell are in polygonal or short fusiformis, and endochylema is less, and caryoplasm ratio is big, and kernel is apparent, and part cell is in into threadiness；2, serious immune rejection problems not will produce；3, convenient material drawing safety and cross-infection risk are small, can be used for repair deficiency tooth and regeneration of tooth；4, it can promote skin wound healing and regeneration, slow down aging or cure blindness, in addition, can also to treat heart disease, rheumatoid arthritis, burn, apoplexy or cartilage impaired etc. for dental pulp stem cell, it is widely used.
Description
Technical field
The present invention relates to biomedical engineering field, more particularly to a kind of cultural method of dental pulp mescenchymal stem cell.
Background technology
1999,《Science》HESC's achievement in research is chosen as first of ten big Progress ＆ New Products of the current year world,
2000,《Time》Weekly is classified as first of ten big technological achievement of the world at the end of the 20th century, and thinks embryonic stem cell and the mankind
Genome will most develop as the new century simultaneously and the field of application prospect.So far stem-cell research becomes biology in recent years
And one of most noticeable hot spot in medical domain.Stem cell is that " trunk " work is played in the growth and development of bion
Initial cell, is a kind of cell colony with self-renewing, hyperproliferation and multi-lineage potential, it includes that embryo is dry
Cell and adult stem cell.Although embryonic stem cell can be divided into various cell types, this differentiation is " non-locating " "
, and there are ethics etc..Ethics, law, immunological rejection etc. is then not present in adult stem cell
Problem, while having many advantages, such as that convenient material drawing, source are wide, thus, adult stem cell is in organizational project, gene therapy and individuation
There is preferable potential applicability in clinical practice in the research for the treatment of.
In fact, the source of stem cell is very extensive.Including deciduous teeth, marrow, Cord blood, umbilical cord, wisdom tooth etc., but different tissues
Stem cell using when be distinguishing.Main product is that Cord blood, umbilical cord spread out the stem cell storage stretched currently on the market, though
So wherein contain abundant stem cell, but be confined to being collected at that moment to realize when delivery of fetus goes out, preserves.And deciduous teeth tooth
The activity of marrow stem cell is 3 times of stem cell, and can Multidirectional Differentiation at groups such as connective tissue, nerve, bone, muscle, teeth
Cell is knitted, application prospect is boundless.
Pulp tissue is located in the pulp cavity of Its pulp, is unique soft tissue in tissue of tooth.Through research, scientist
It is found that a kind of and mesenchymal stem cell has extremely similar immunophenotype and forms the cell of Mineral nodules ability, carefully
In born of the same parents form be in fusiformis, can self-renewing and Multidirectional Differentiation, have stronger cloning capacity.These in pulp tissue by isolating
Be known as dental pulp stem cell at fibrous cell.
" every tooth all includes abundant stem cell ' resource ', is stored in health, is having illness needs in the future
Shi Peiyu breaks up stem cell, it might even be possible to save somebody's life.”
China reaches underpopulated the 1% of World Health Organization's dental health standard, 17 years old or less person, missing one or more
The people of the case where polydonita tooth accounts for 7%, and the age is more than 50 years old person, and average agomphosis number is 12, general once receiving port intracavitary of being grown up
Dental prosthesis accounts for about 85%.
Healthy seed is preserved, since storing dental pulp stem cell, there is self-renewing energy present in the pulp tissue of people
The adult stem cell of power, exuberant ability, multiphase differentiation potential, i.e. dental pulp stem cell (Dental pulp stem cells,
FPSCs)。
Dental pulp stem cell belongs to mescenchymal stem cell, and is the strongest mescenchymal stem cell of activity, active marrow stem
3 times of cell.A small amount of dental pulp stem cell can divide out thousands upon thousands cells.Differentiation capability is very strong, it is suitable in vivo or
It can break up under vitro as a variety of human body cells such as osteocyte, adipocyte and liver cell, for tissue, organ reparation
New cell origin is provided with transplanting.
Dental pulp stem cell can keep itself undifferentiated state and proliferative capacity and be divided under the suitable conditions
Multiple functions cell or tissue organ, such as periodontium, nerve, muscle and adipocyte etc. are perplexed to be deep by odontopathy
Or bring new hope for its worried people, and as disease research range expands, future diabetes, spinal cord injury,
Apoplexy, valvulopathy, periodontosis etc. are all expected to obtain medical treatment using dental pulp stem cell.
Currently, common technology is by enzyme digestion original cuiture dental pulp stem cell, the people's dental pulp turned out in this way is dry
The adherent slow, growth of cell is slowly.4h adherent about 70%.12h starts to stretch.48h is unfolded substantially.Cell in it is most of for spindle shape at
Threadiness, 1~2 protrusion, minority are polygonal, fusiform or ellipse, and small volume cell spaces are plentiful, and about 10~12d reaches
To 90%.
Invention content
It is described above in order to overcome the shortcomings of, the object of the present invention is to provide a kind of convenient material drawing safety, immunological rejection and
Cross-infection risk is small, for repair deficiency tooth and regeneration of tooth, promote skin wound healing and regeneration, slow down aging, control
More it blinds, treat the dental pulp mescenchymal stem cells of the diseases such as heart disease, rheumatoid arthritis, burn, apoplexy or cartilage be impaired
Cultural method.
The present invention the technical solution to solve the technical problem is that：A kind of cultural method of dental pulp mescenchymal stem cell, including
Following steps：
S1, prepare dental pulp sample：By tooth samples be immersed in buffer solution or by tooth samples it is stored refrigerated, preserve when
Between in 40h~47h, corona of riving thereafter takes out dental pulp；
S2, it is separately cultured dental pulp mescenchymal stem cell:After being rinsed with 0.01M frozen stock solutions PBS, the dental pulp of taking-up is cut into about
1mm3×1mm3×1mm3Fritter, then 1h is digested in 37 DEG C with 0.3% collagenase type I and 0.4% catabolic enzyme, gently blow
Discrete unicellular agglomerate is beaten, the single cell suspension of formation is centrifuged by aperture for 800r/min after 70 μm of cell screen clothes
5min；
0.01M frozen stock solutions PBS is used to rinse again primary, by cell precipitation, while by density 5000/cm2Stem cell culture
Base is inoculated in culture bottle and cultivates, and liquid is changed 1 time per 3d；
S3, colonized culture detach dental pulp mescenchymal stem cell：When cell growth is up to 80%~85% degree of converging, use
0.25% pancreatin had digestive transfer culture, then rinsed with 0.01M frozen stock solutions PBS it is primary, by cell precipitation, while by density 5000/cm2
Stem cell media be inoculated in culture bottle and cultivate, change liquid 1 time per 3d, when medium cell growth is up to 80% degree of converging, use
0.25% pancreatin digestion harvest；
S4, the detection of dental pulp mescenchymal stem cell：By the filial generation dental pulp mescenchymal stem cell of harvest, cell is resuspended using PBS,
Adjustment cell density is 1 × 106/ml, takes out 1ml, and the absolute ethyl alcohol 2ml piping and druming mixing of precooling is added, at 4 DEG C overnight and from
The heart, which is abandoned after ethyl alcohol, carries out cell cycle detection on flow cytometer again；
S5, the detection dyeing of dental pulp mescenchymal stem cell.
In this embodiment, the tooth samples are 19-29 years old complete third molars of health adult's impaction, and
Tooth samples impregnate and it is stored refrigerated before need to dental surface carry out sterilization treatment.
In this embodiment, the depth that corona is rived is 0.6mm~0.9mm, and the depth rived using corona,
Under aseptic condition, pulp tissue is exposed, takes out dental pulp later.
In this embodiment, the operation tool for taking out dental pulp is any of which of root canal file, curet, tweezers and probe
Kind.
In this embodiment, α-MEM containing 15% fetal calf serum in the stem cell media, 5%
DMEM culture mediums and 5% serum free medium.
In this embodiment, in S4, it is also necessary to be rinsed twice with 0.01M frozen stock solutions PBS, 0.5% PI is used in combination
30min is dyed in 4 DEG C.
In this embodiment, in S5, well-grown colonized culture cell is taken to do cell climbing sheet, with LSAB methods
Anti- Vimemin, CD44, ON, DSP immunohistochemical staining of row.
In this embodiment, further include the steps that filtration sterilization after the centrifugation in S2 and S4.
In this embodiment, the culture bottle is rolling bottle, bioreactor or T75 culture bottles
In this embodiment, the conditioned medium of the FBS and dental pulp mescenchymal stem cell is referred to than being 4：5.
The beneficial effects of the present invention are：
1, close using being arranged between the cell of clone and separate culture, central part cell boundary is unclear.It is rounded or do not advise
Then shape and formation cellular nodules sample, peripheral portion cell are in polygonal or short fusiformis, and endochylema is less, and caryoplasm ratio is big, and kernel is apparent,
Part cell is in and to cultivate 3 days and cover at threadiness；
2, dental pulp mescenchymal stem cell be by it is personal oneself tissue it is separated and turn out, be reused in self
On, so not will produce serious immune rejection problems in treatment；
3, convenient material drawing safety and cross-infection risk are small, are different from cord blood stem cell, so medical value is very big, it can
For repair deficiency tooth and regeneration of tooth；
4, skin wound healing and regeneration can be promoted, slow down aging or cure blindness, in addition, dental pulp mescenchymal stem cell is also
It is impaired etc. that heart disease, rheumatoid arthritis, burn, apoplexy or cartilage can be treated, it is widely used.
Description of the drawings
The present invention is described in detail by following preferred embodiments and attached drawing for ease of explanation,.
Fig. 1 is the concrete operations flow chart of the present invention；
Fig. 2 is the schematic diagram that the present invention can treat kinds of Diseases.
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.
It should be noted that in the absence of conflict, the feature in embodiment and embodiment in the present invention can phase
Mutually combination.
In the description of the present invention, it should be noted that unless otherwise clearly defined and limited, term " installation ", " phase
Even ", " connection " shall be understood in a broad sense, for example, it may be being fixedly connected, may be a detachable connection, or be integrally connected；It can
Can also be electrical connection to be mechanical connection；It can be directly connected, can also indirectly connected through an intermediary, Ke Yishi
Connection inside two elements.For the ordinary skill in the art, above-mentioned term can be understood by concrete condition
Concrete meaning in the present invention.
Embodiment one
As shown in Fig. 1~2, a kind of cultural method of dental pulp mescenchymal stem cell of the invention includes the following steps：
S1, prepare dental pulp sample：By tooth samples be immersed in buffer solution or by tooth samples it is stored refrigerated, preserve when
Between in 40h~47h, corona of riving, the depth that corona is rived is 0.6mm~0.9mm, and the depth rived using corona, in nothing
Under the conditions of bacterium, pulp tissue is exposed, takes out dental pulp later.In this embodiment, tooth samples are 19-29 years old health
Be grown up the complete third molar of impaction, and tooth samples impregnate and it is stored refrigerated before need to carry out at sterilizing dental surface
Reason.In addition, dental pulp stem cell most easily successfully acquires the deciduous teeth and wisdom tooth of source unsoundness.20 deciduous teeth can be used in extraction tooth
The child of marrow stem cell, each dental transitional period is Zhi get ed depositing；If wish of the adult if any extraction of wisdom tooth, tooth can be also carried out
Marrow stem cell stores.Since oral bacteria environment is complicated, dental pulp stem cell is preserved ahead of time, or the success rate that will improve its preparation.
Meanwhile buffer solution can be the buffer solution containing antibiotic, antibiotic can be penicillin or streptomysin.It is specific real at this
It applies in example, the operation tool for taking out dental pulp is any of which of root canal file, curet, tweezers and probe.
S2, it is separately cultured dental pulp mescenchymal stem cell:After being rinsed with 0.01M frozen stock solutions PBS, the dental pulp of taking-up is cut into about
1mm3×1mm3×1mm3Fritter, then with 0.3% collagenase type I and 0.4% catabolic enzyme digestion is carried out in 37 DEG C of water-baths
1h gently blows and beats discrete unicellular agglomerate, after the cell screen clothes for being 70 μm by aperture by the single cell suspension of formation, and with
The centrifugal speed of 800r/min centrifuges 5min；0.01M frozen stock solutions PBS is used to rinse again primary, by cell precipitation, while by density
5000/cm2Stem cell media be inoculated in culture bottle and cultivate, culture medium changes once every three days.In this embodiment,
α-MEM containing 15% fetal calf serum in the stem cell media, 5% DMEM culture mediums and 5% serum free medium.
The conditioned medium of FBS and dental pulp mescenchymal stem cell is referred to than being 4：5.
S3, colonized culture detach dental pulp mescenchymal stem cell：When cell growth is up to 80%~85% degree of converging, use
0.25% pancreatin had digestive transfer culture, then rinsed with 0.01M frozen stock solutions PBS it is primary, by cell precipitation, while by density 5000/cm2
Stem cell media be inoculated in T75 culture bottles and cultivate, change liquid 1 time per 3d, medium cell growth is up to 80% degree of converging
When, digest harvest with 0.25% pancreatin.
S4, the detection of dental pulp mescenchymal stem cell：By the filial generation dental pulp mescenchymal stem cell of harvest, cell is resuspended using PBS,
Adjustment cell density is 1 × 106/ml, takes out 1ml, and the absolute ethyl alcohol 2ml piping and druming mixing of precooling is added, at 4 DEG C overnight and from
The heart, which is abandoned after ethyl alcohol, carries out cell cycle detection on flow cytometer again, detect the specificity of cell, including mescenchymal stem cell is special
Anisotropic surface marker Stro-1, CD29, CD44, CD71, CD90, CD106, CD120 etc. and other system's cell surface marks
Will object, such as CD14, CD34, CD45.In this embodiment, it is also necessary to be rinsed twice, be used in combination with 0.01M frozen stock solutions PBS
0.5% PI dyes 30min in 4 DEG C.
S5, the detection dyeing of dental pulp mescenchymal stem cell.In S5, takes well-grown colonized culture cell to do cell and climb
Piece, with anti-Vimemin, CD44, ON, DSP immunohistochemical staining of LSAB method rows.
In this embodiment, further include the steps that filtration sterilization after the centrifugation in S2 and S4.
Close using being arranged between the cell of clone and separate culture, central part cell boundary is unclear.It is rounded or irregular
Shape and formation cellular nodules sample, peripheral portion cell are in polygonal or short fusiformis, and endochylema is less, and caryoplasm ratio is big, and kernel is apparent, portion
Point cell is in and to cultivate 3 days and cover at threadiness.
Embodiment two
A kind of cultural method of dental pulp mescenchymal stem cell of the present invention, includes the following steps：
S1, prepare dental pulp sample：By tooth samples be immersed in buffer solution or by tooth samples it is stored refrigerated, preserve when
Between in 40h~47h, corona of riving, the depth that corona is rived is 0.6mm~0.9mm, and the depth rived using corona,
Under aseptic condition, pulp tissue is exposed, takes out dental pulp later.In this embodiment, tooth samples are 19-29 years old strong
Health be grown up the complete third molar of impaction, and tooth samples impregnate and it is stored refrigerated before need to carry out at sterilizing dental surface
Reason.In addition, dental pulp stem cell most easily successfully acquires the deciduous teeth and wisdom tooth of source unsoundness.20 deciduous teeth can be used in extraction tooth
The child of marrow stem cell, each dental transitional period is Zhi get ed depositing；If wish of the adult if any extraction of wisdom tooth, tooth can be also carried out
Marrow stem cell stores.Since oral bacteria environment is complicated, dental pulp stem cell is preserved ahead of time, or the success rate that will improve its preparation.
Meanwhile buffer solution can be the buffer solution containing antibiotic, antibiotic can be penicillin or streptomysin.It is specific real at this
It applies in example, the operation tool for taking out dental pulp is any of which of root canal file, curet, tweezers and probe.
S2, it is separately cultured dental pulp mescenchymal stem cell:After being rinsed with 0.01M frozen stock solutions PBS, the dental pulp of taking-up is cut into about
1mm3×1mm3×1mm3Fritter, then with 0.3% collagenase type I and 0.4% catabolic enzyme digestion is carried out in 37 DEG C of water-baths
1h gently blows and beats discrete unicellular agglomerate, after the cell screen clothes for being 70 μm by aperture by the single cell suspension of formation, and with
The centrifugal speed of 800r/min centrifuges 5min；0.01M frozen stock solutions PBS is used to rinse again primary, by cell precipitation, while by density
5000/cm2Stem cell media be inoculated in culture bottle and cultivate, culture medium changes once every three days.In this embodiment,
α-MEM containing 15% fetal calf serum in the stem cell media, 5% DMEM culture mediums and 5% serum free medium.
The conditioned medium of FBS and dental pulp mescenchymal stem cell is referred to than being 4：5.
S3, colonized culture detach dental pulp mescenchymal stem cell：When cell growth is up to 80%~85% degree of converging, use
0.25% pancreatin had digestive transfer culture, then rinsed with 0.01M frozen stock solutions PBS it is primary, by cell precipitation, while by density 5000/cm2
Stem cell media be inoculated in bioreactor and cultivate, change liquid 1 time per 3d, medium cell growth is up to 80% degree of converging
When, digest harvest with 0.25% pancreatin.Bioreactor culture amplification can be level-one bioreactor culture, can also be
Multistage culture.In level-one bioreactor cell growth to a certain extent when, can be carried out down according to the requirement to cell quantity
The amplification culture of level-one cell will be inoculated with next stage biological respinse after the cell on microcarrier completely digestion as seed cell
Device directly adds new microcarrier and carries out ball and turns ball amplification culture.
Specifically, bioreactor can be selected from stirring type bioreactor, fixed-bed bioreactor, wave (wave)
Bioreactor etc. and other various forms and cells in vitro Large-scale culture systems or device based on various principles.It stirs
It mixes formula bioreactor and culture solution is mainly stirred to increase mass transfer ability by blender rotation, it is ensured that the oxygen of cell culture
The homogeneity of concentration and nutriment achievees the purpose that large-scale culture cell, while culture solution being made to apply centainly cell
Shear stress.The most commonly used is blender jar reactors in medical tissue engineering research at present.It is mainly characterized by turning using magnetic force
Son agitation culture solution, stem cell is seeded in the culture solution of magnetic agitation, cell is grown in the culture solution of prolonged agitation.It stirs
Mixing the great advantage of formula bioreactor is, can cultivate various types of zooblasts, and culture process is easy amplification, product matter
Amount is stablized, and is very suitable for being factory produced.Chip carrier is filled inside fixed-bed bioreactor, and cell docile is made to grow,
So that cell is reached higher density by continuous pouring fresh medium, in incubation, cytotrophy is sufficient, metabolic waste energy and
When removal for cell growth provide a good environment.The chip carrier used include polyester fiber of NBS companies etc. all
It can be in favor of the material of cell docile growth.WAVE bioreactors are disposable wave bag bioreactors, to
Substitute traditional stainless steel fermentation tank.One-time reaction device is suitable for various types of cell culture.Mild undulation
Good oxygen supply and mixed purpose can be played, meanwhile, the also very little of shearing force caused by this motion mode, far smaller than
The shearing force caused by stirring or gas-lifting type method in traditional tank body；It can support high density mass cell culture, and
And the destruction of foam and shearing force will not be formed.
S4, the detection of dental pulp mescenchymal stem cell：By the filial generation dental pulp mescenchymal stem cell of harvest, cell is resuspended using PBS,
Adjustment cell density is 1 × 106/ml, takes out 1ml, and the absolute ethyl alcohol 2ml piping and druming mixing of precooling is added, at 4 DEG C overnight and from
The heart, which is abandoned after ethyl alcohol, carries out cell cycle detection on flow cytometer again, detect the specificity of cell, including mescenchymal stem cell is special
Anisotropic surface marker Stro-1, CD29, CD44, CD71, CD90, CD106, CD120 etc. and other system's cell surface marks
Will object, such as CD14, CD34, CD45.In this embodiment, it is also necessary to be rinsed twice, be used in combination with 0.01M frozen stock solutions PBS
0.5% PI dyes 30min in 4 DEG C.
S5, the detection dyeing of dental pulp mescenchymal stem cell.In S5, takes well-grown colonized culture cell to do cell and climb
Piece, with anti-Vimemin, CD44, ON, DSP immunohistochemical staining of LSAB method rows.
In this embodiment, further include the steps that filtration sterilization after the centrifugation in S2 and S4.
Close using being arranged between the cell of clone and separate culture, central part cell boundary is unclear.It is rounded or irregular
Shape and formation cellular nodules sample, peripheral portion cell are in polygonal or short fusiformis, and endochylema is less, and caryoplasm ratio is big, and kernel is apparent, portion
Point cell is in and to cultivate 3 days and cover at threadiness.
Embodiment three
The cultural method of another dental pulp mescenchymal stem cell of the present invention, includes the following steps：
S1, prepare dental pulp sample：By tooth samples be immersed in buffer solution or by tooth samples it is stored refrigerated, preserve when
Between in 40h~47h, corona of riving, the depth that corona is rived is 0.6mm~0.9mm, and the depth rived using corona,
Under aseptic condition, pulp tissue is exposed, takes out dental pulp later.In this embodiment, tooth samples are 19-29 years old strong
Health be grown up the complete third molar of impaction, and tooth samples impregnate and it is stored refrigerated before need to carry out at sterilizing dental surface
Reason.In addition, dental pulp stem cell most easily successfully acquires the deciduous teeth and wisdom tooth of source unsoundness.20 deciduous teeth can be used in extraction tooth
The child of marrow stem cell, each dental transitional period is Zhi get ed depositing；If wish of the adult if any extraction of wisdom tooth, tooth can be also carried out
Marrow stem cell stores.Since oral bacteria environment is complicated, dental pulp stem cell is preserved ahead of time, or the success rate that will improve its preparation.
Meanwhile buffer solution can be the buffer solution containing antibiotic, antibiotic can be penicillin or streptomysin.It is specific real at this
It applies in example, the operation tool for taking out dental pulp is any of which of root canal file, curet, tweezers and probe.
S2, it is separately cultured dental pulp mescenchymal stem cell:After being rinsed with 0.01M frozen stock solutions PBS, the dental pulp of taking-up is cut into about
1mm3×1mm3×1mm3Fritter, then with 0.3% collagenase type I and 0.4% catabolic enzyme digestion is carried out in 37 DEG C of water-baths
1h gently blows and beats discrete unicellular agglomerate, after the cell screen clothes for being 70 μm by aperture by the single cell suspension of formation, and with
The centrifugal speed of 800r/min centrifuges 5min；0.01M frozen stock solutions PBS is used to rinse again primary, by cell precipitation, while by density
5000/cm2Stem cell media be inoculated in culture bottle and cultivate, culture medium changes once every three days.In this embodiment,
α-MEM containing 15% fetal calf serum in the stem cell media, 5% DMEM culture mediums and 5% serum free medium.
The conditioned medium of FBS and dental pulp mescenchymal stem cell is referred to than being 4：5.
S3, colonized culture detach dental pulp mescenchymal stem cell：When cell growth is up to 80%~85% degree of converging, use
0.25% pancreatin had digestive transfer culture, then rinsed with 0.01M frozen stock solutions PBS it is primary, by cell precipitation, while by density 5000/cm2
Stem cell media be inoculated in rolling bottle and cultivate, change liquid 1 time per 3d, when medium cell growth is up to 80% degree of converging, use
0.25% pancreatin digestion harvest.
In the present embodiment, the spinner culture cell containing microcarrier is used, rolling bottle working volume is 125ml, micro-
Carrier is Cytodex-3.
In the present embodiment, S3 is as follows：
A, rolling bottle pre-processes：125ml rolling bottles clean up, and are dried to and are completely dried, and carry out sterilization treatment, and silication is added
For liquid 15ml~18ml in rolling bottle, slowly rotating rolling bottle makes silication liquid infiltrate bottle wall, and after sucking silication liquid, rolling bottle is placed in ventilation
Place air-dries 15h, and distilled water flushing is spare；
B, microcarrier pre-processes：Microcarrier density is set to 2g/L in the present embodiment, weighs 0.05gCytodex-3 additions and turns
In bottle, 0.01M frozen stock solutions PBS is added and impregnates 3h, the new PBS of 20ml are added after sucking, 130 DEG C of high pressure steam sterilizations suck
PBS, and the α-MEM culture solutions that 20ml contains 17% fetal calf serum, 37 DEG C of soaked overnights are added.
C, it changes liquid 1 time daily, when medium cell growth is up to 80% degree of converging, digests harvest with 0.25% pancreatin.
S4, the detection of dental pulp mescenchymal stem cell：By the filial generation dental pulp mescenchymal stem cell of harvest, cell is resuspended using PBS,
Adjustment cell density is 1 × 106/ml, takes out 1ml, and the absolute ethyl alcohol 2ml piping and druming mixing of precooling is added, at 4 DEG C overnight and from
The heart, which is abandoned after ethyl alcohol, carries out cell cycle detection on flow cytometer again, detect the specificity of cell, including mescenchymal stem cell is special
Anisotropic surface marker Stro-1, CD29, CD44, CD71, CD90, CD106, CD120 etc. and other system's cell surface marks
Will object, such as CD14, CD34, CD45.In this embodiment, it is also necessary to be rinsed twice, be used in combination with 0.01M frozen stock solutions PBS
0.5% PI dyes 30min in 4 DEG C.
S5, the detection dyeing of dental pulp mescenchymal stem cell.In S5, takes well-grown colonized culture cell to do cell and climb
Piece, with anti-Vimemin, CD44, ON, DSP immunohistochemical staining of LSAB method rows.
In this embodiment, further include the steps that filtration sterilization after the centrifugation in S2 and S4.
Close using being arranged between the cell of clone and separate culture, central part cell boundary is unclear.It is rounded or irregular
Shape and formation cellular nodules sample, peripheral portion cell are in polygonal or short fusiformis, and endochylema is less, and caryoplasm ratio is big, and kernel is apparent, portion
Point cell is in and to cultivate 3 days and cover at threadiness.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement etc., should all be included in the protection scope of the present invention made by within refreshing and principle.
Claims (10)
1. a kind of cultural method of dental pulp mescenchymal stem cell, which is characterized in that include the following steps：
S1, prepare dental pulp sample：By tooth samples be immersed in buffer solution or by tooth samples it is stored refrigerated, the holding time exists
40h ~ 47h, corona of riving thereafter take out dental pulp;
S2, it is separately cultured dental pulp mescenchymal stem cell:After being rinsed with 0.01M frozen stock solutions PBS, the dental pulp of taking-up is cut into about 1mm3
×1mm3×1mm3Fritter, then 1h is digested in 37 DEG C with 0.3% collagenase type I and 0.4% catabolic enzyme, it gently blows and beats discrete
The single cell suspension of formation is centrifuged 5min by unicellular agglomerate by aperture for 800r/min after 70 μm of cell screen clothes；
0.01M frozen stock solutions PBS is used to rinse again primary, by cell precipitation, while by density 5000/cm2Stem cell media inoculation
It is cultivated in culture bottle, liquid is changed 1 time per 3d；
S3, colonized culture detach dental pulp mescenchymal stem cell：When cell growth is up to 80% ~ 85% degree of converging, with 0.25%
Pancreatin had digestive transfer culture, then rinsed with 0.01M frozen stock solutions PBS it is primary, by cell precipitation, while by density 5000/cm2Stem cell
Culture medium inoculated is cultivated in culture bottle, liquid is changed 1 time per 3d, when medium cell growth is up to 80% degree of converging, with 0.25% pancreas
Enzymic digestion harvests；
S4, the detection of dental pulp mescenchymal stem cell：By the filial generation dental pulp mescenchymal stem cell of harvest, cell, adjustment is resuspended using PBS
Cell density is 1 × 106/ml, takes out 1ml, and the absolute ethyl alcohol 2ml piping and druming mixings of precooling are added, stays overnight and centrifuges at 4 DEG C and abandon
Carry out cell cycle detection after ethyl alcohol on flow cytometer again；
S5, the detection dyeing of dental pulp mescenchymal stem cell.
2. the cultural method of dental pulp mescenchymal stem cell according to claim 1, which is characterized in that the tooth samples are
19-29 years old complete third molars of health adult's impaction, and tooth samples impregnate and it is stored refrigerated before need to dental surface
Carry out sterilization treatment.
3. the cultural method of dental pulp mescenchymal stem cell according to claim 1, which is characterized in that the depth that corona is rived
For 0.6mm ~ 0.9mm, and the depth rived using corona is aseptically exposed pulp tissue, takes out dental pulp later.
4. the cultural method of dental pulp mescenchymal stem cell according to claim 1, which is characterized in that take out the operation of dental pulp
Tool is any of which of root canal file, curet, tweezers and probe.
5. the cultural method of dental pulp mescenchymal stem cell according to claim 1, which is characterized in that the stem cell culture
α-MEM containing 15% fetal calf serum in base, 5% DMEM culture mediums and 5% serum free medium.
6. the cultural method of dental pulp mescenchymal stem cell according to claim 1, which is characterized in that in S4, it is also necessary to
It is rinsed twice with 0.01M frozen stock solutions PBS, the PI of 0 .5% is used in combination to dye 30min in 4 DEG C.
7. the cultural method of dental pulp mescenchymal stem cell according to claim 1, which is characterized in that in S5, take growth
Good colonized culture cell does cell climbing sheet, with anti-Vimemin, CD44, ON, DSP immunohistochemical staining of LSAB method rows.
8. the cultural method of dental pulp mescenchymal stem cell according to claim 1, which is characterized in that in S2 and S4 from
It further include the steps that filtration sterilization after the heart.
9. the cultural method of dental pulp mescenchymal stem cell according to claim 1, which is characterized in that the culture bottle is to turn
Bottle, bioreactor or T75 culture bottles.
10. the cultural method of dental pulp mescenchymal stem cell according to claim 1, which is characterized in that the FBS and dental pulp
The conditioned medium of mescenchymal stem cell is referred to than being 4：5.
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