CN107267451A - A kind of preparation method of primary dental pulp stem cell and the method for building dental pulp stem cell storehouse - Google Patents
A kind of preparation method of primary dental pulp stem cell and the method for building dental pulp stem cell storehouse Download PDFInfo
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- CN107267451A CN107267451A CN201710626703.9A CN201710626703A CN107267451A CN 107267451 A CN107267451 A CN 107267451A CN 201710626703 A CN201710626703 A CN 201710626703A CN 107267451 A CN107267451 A CN 107267451A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
A kind of preparation method of primary dental pulp stem cell and the method for building dental pulp stem cell storehouse, using constant-temperature table under specified temp and speed conditions, pulp tissue can be digested in a few minutes clock time, extract enough dental pulp stem cells with preferable biological activity, both the time of the primary extraction of dental pulp stem cell had been shortened, time cost is saved, it can mass produce, the enough dental pulp stem cells obtained simultaneously, it disclosure satisfy that dental pulp stem cell in the structure of stem cell bank and the use demand of later stage stem cell clinical treatment and regenerative medicine research field, compared with pulp tissue under normal condition half digests 30min or totally disappeared 1h or so control group, DPSCs amounts are without obvious significant difference, but cell-proliferation activity is apparently higher than control group.Therefore, this method can be carried out quickly in batch in the building process in DPSCs storehouses, shortened the time of the primary extractions of DPSCs, saved time cost, be suitable for large-scale production.
Description
Technical field
Present invention relates particularly to technical field of stem cell culture, and in particular to a kind of preparation method of primary dental pulp stem cell
And the method for building dental pulp stem cell storehouse.
Background technology
Stem cell is the cell that a class has self-renewing, hyperproliferation and Multidirectional Differentiation ability, under given conditions can
Induction differentiation is carried out to human body mature tissue, organ, has been widely used in the research of regenerative medicine various aspects.Stem cell
Source is mainly embryonic stem cell and mescenchymal stem cell, and wherein embryonic stem cell limits it and faced due to being related to ethnics Problem
Bed Study on Transformation；Mescenchymal stem cell main source marrow, i.e. mesenchymal stem cells MSCs, but it is in clinical Study on Transformation process
It is middle to there is the shortcomings of causing invasive damage to donor.Since Gronthos in 2000 etc. [GRONTHOS S, MANKANI M,
BRAHIM J, et al.Postnatal human dental pulp stem cells (DPSCs) in vitro and in
Vivo.Proc Natl Acad Sci USA, 2000,97 (25)：13625-13630.] found from health adult's third molar
Since dental pulp stem cell (dental pulp stem cells, DPSCs), in the past as the tooth that comes off of oral medical discarded object
" turn waste into wealth " with extraction of wisdom tooth etc., the main source studied as DPSCs.DPSCs is a kind of with hyperproliferation energy
The mescenchymal stem cell of power and multi-lineage potential, with abundance, materials safe ready, ethnics Problem is not related to, exempts from
The low advantage of epidemic focus, has made great progress in regenerative medicine field, has broad application prospects.Meanwhile, DPSCs is suitable
The Various Tissues such as bone tissue, cartilaginous tissue, adipose tissue, nerve fiber, muscular tissue, cornea can be induced to differentiate under the conditions of
Cell, and can be induced as induced multi-potent stem cell (the induced pluripotent with embryonic stem cell-like characteristic
Stem cells, iPSCs), can be as the seed cell in later a variety of Disease Clinical Study on Transformation.At present, it is existing multiple
Country sets up dental pulp stem cell storehouse for clinical treatment or scientific research needs.
It is dry thin that the building process in dental pulp stem cell storehouse includes dental pulp stem cell culture, the passage of dental pulp stem cell and dental pulp
Born of the same parents freeze three parts.Wherein, the incubation of dental pulp stem cell be divided into again tooth body collect, tooth body transport, pulp tissue extract with
And the culture of stem cell etc., and each step in this process all have to the final biological activity for obtaining dental pulp stem cell compared with
Big influence.At present, the extraction for pulp tissue and incubation, conventional method are the direct cultivation of tissue block and clostridiopetidase A
Digest two kinds of cultivation, but have that cultivation cycle is long, cell concentration is less, be easily caused differentiation and digestion not exclusively or digested
The shortcomings of spending.
The time that traditional enzyme digestion prepares dental pulp stem cell digestion is 20min-3h, such as patent type I collagen
Mentioned inside the construction method (A of CN 101717750) in storehouse, the pulp tissue originated from correction or wisdom tooth utilizes collagen after shredding
Enzyme and dispase are in 37 DEG C, 5%CO250min is digested in environment and obtains dental pulp stem cell；A kind of clinic dental pulp stem cell of patent
And preparation method thereof (A of CN 104694464) pulp tissue digestion 1-3h is obtained into dental pulp using clostridiopetidase A and dispase II
Stem cell；Patent is a kind of to prepare dental pulp mescenchymal stem cell and the method for setting up dental pulp mesenchyma stem cell and cell recovery side
Method (A of CN 104630141) obtains dental pulp stem cell using pancreatin substitute digestion pulp tissue 20min.
The content of the invention
In order to overcome the defect that above-mentioned prior art is present, there is provided a kind of preparation method of primary dental pulp stem cell and structure
The method in dental pulp stem cell storehouse, pulp tissue (≤10min) can be digested, extract enough in a short time
DPSCs。
The technical solution that the present invention is used is：A kind of preparation method of primary dental pulp stem cell, comprises the following steps：
(1) tooth body is collected；
(2) dental pulp is extracted：Dental pulp is extracted after the Isolated Tooth of collection is carried out into pre-treatment；
(3) pulp tissue's digestion and stem cell culture：The pulp tissue of extraction is placed in sterile PBS, dental pulp group is shredded
Knit to 1mm2, then, pulp tissue is collected using centrifuge tube, and in 1000rpm under normal temperature condition, centrifuge 10min, abandon supernatant,
3mg/mL type i collagen enzyme+4mg/mL neutral proteinase mixed enzymes are added dropwise and dispel pulp tissue, type i collagen enzyme and neutral proteinase
Volume ratio is 1: 1, is placed in 37 DEG C, 180rpm constant-temperature table environment and is digested, after 5-10min, and blood serum medium is added dropwise
Terminate under the conditions of digestion, 1000rpm, centrifugal treating 5min collects tissue block and cell, the complete α-MEM culture mediums of 1mL are resuspended, connect
Plant in 35mm sterile petri dish, in 37 DEG C, 5%CO2Cultivated in cell culture incubator, complete α-MEM culture mediums after 1d
Fluid infusion 2mL, a α-MEM culture medium is changed every 3d；
(4) passage processing is carried out to dental pulp stem cell.
Described complete α-MEM culture mediums are 10-20%FBS+100U/mL penicillin+100ug/mL streptomysins.
Pre-treatment is the tooth body 2 times that Isolated Tooth is rinsed using tooth storing liquid during described step (2) dental pulp is extracted, every time
1min, then steeps Isolated Tooth 1min using 1% Povidone Iodine Disinfection Solution, finally rinses Isolated Tooth 2 times with tooth storing liquid again, often
Secondary 1min.
Described step (4) carries out passage process step to dental pulp stem cell：When pulp cells fusion reaches 80%-
When 90%, digested with pancreatin/EDTA mixed enzymes, 5min is centrifuged under the conditions of 1000rpm, collect cell, trained with complete α-MEM
Base cell dispersion is supported, cell suspension is evenly distributed in 2 Tissue Culture Dish, is placed in 37 DEG C, 5%CO2Trained in incubator
Support, liquid is changed once every 3d.
A kind of method for building dental pulp stem cell storehouse, comprises the following steps：
(1) tooth body is collected；
(2) tooth body is transported；
(3) dental pulp is extracted；
(4) pulp tissue's digestion and stem cell culture；
(5) passage of dental pulp stem cell；
(6) the biological function detection of dental pulp stem cell；
(7) dental pulp stem cell freezes and recovered.
Described dental pulp stem cell biological function detection include CCK-8 detection, immunofluorescence dyeing detect, into fat to
The one or more of differentiation detection, skeletonization to differentiation detection or into cartilage into differentiation detection.
Described freezing for step (7) dental pulp stem cell comprises the following steps：Take the dental pulp for meeting standard logarithmic growth period
Stem cell, when cell fusion reaches 80%-90%, is digested with pancreatin/EDTA mixed enzymes, is centrifuged under the conditions of 1000rpm
5min, collects cell, and with 10%DMSO+90%FBS cells frozen storing liquid, adjustment cell concentration is 3 × 106Individual/mL, by cell
Suspension 1mL is respectively charged into cryopreservation tube, and then cryopreservation tube number consecutively is put into the special freezing storing box of cell, with 1 DEG C/min speed
Degree cooling, is put into -80 DEG C of refrigerators and freezes 12h, last cell cryopreservation tube is transferred in liquid nitrogen to be stored for a long time, and carries out corresponding
Registration.
The recovery of described step (7) dental pulp stem cell comprises the following steps：After cell cryopreservation tube takes out from liquid nitrogen, soon
Speed, which is put into 37 DEG C of water baths, is thawed, and is then diluted with 5mL complete α-MEM culture mediums, under the conditions of 1000rpm
5min is centrifuged, cell is collected, is dispelled with complete α-MEM culture mediums, be transferred in 35-mm steril cell culture dishes, be put into 37 DEG C,
5%CO2Cultivated in cell culture incubator, change a subculture within 3 days.
The beneficial effects of the invention are as follows：The invention provides a kind of preparation method of primary dental pulp stem cell and structure dental pulp
The method of stem cell bank, can be in a few minutes clock time by dental pulp group using constant-temperature table under specified temp and speed conditions
Knit and digest, extract enough dental pulp stem cells with preferable biological activity, both shortened dental pulp stem cell primary
The time of extraction, time cost is saved, can mass produced, while the enough dental pulp stem cells obtained, disclosure satisfy that tooth
Marrow stem cell in the structure of stem cell bank and the use demand of later stage stem cell clinical treatment and regenerative medicine research field,
Compared with pulp tissue under normal condition half digests 30min or totally disappeared 1h or so control group, DPSCs amounts are poor without obvious statistics
It is different, but cell-proliferation activity is apparently higher than control group.Therefore, this method can be quick in batch in the building process in DPSCs storehouses
Carry out, shorten the time of the primary extractions of DPSCs, saved time cost, be suitable for large-scale production.
Figure of description
Fig. 1 is the extraction flow chart of pulp tissue of the present invention.
Fig. 2 is the light microscopic picture of the first generation after different condition and different time dental pulp stem cell culture 8d and passage,
Wherein TES is represented in constant-temperature table digestion condition, and TE represents half and digested, and E represents totally disappeared.
Fig. 3 is different condition and different time dental pulp stem cell CCK-8 testing results, and wherein TES is represented and shaken in constant temperature
Bed digestion condition, TE represents half and digested, and E represents totally disappeared.
Embodiment
With reference to embodiment, the present invention will be described in detail, and embodiment is only the preferred embodiment of the present invention,
It is not limitation of the invention.
Tooth body is collected
After preengaging and register in advance through patient, specified stomatological hospital clinic collect pull out without the healthy wisdom tooth of dental caries tooth
(age≤30 year old).First, Isolated Tooth is rinsed 2 times using sterile PBS buffer, each 1min；Secondly, it is curved using Sterile ophthalmic
Tweezer carefully clears up the remaining soft tissue of in vitro tooth surface, and the Isolated Tooth 2 times rinsed again with sterile PBS buffer after cleaning,
Each 1min；Finally, Isolated Tooth is wiped 2 times using 75% cotton ball soaked in alcohol, each 1min is subsequently placed in containing 4 DEG C of tooth storages
In the sterilized tooth collecting pipe of liquid, ice chest is preserved.
Tooth storing liquid is configured：Sterile PBS buffer+dual anti-(100U/mL penicillin+100ug/mL streptomysins), volume
Ratio 10: 1.
Tooth body is transported
The Isolated Tooth being collected into is transported to stem cell laboratory and in extracting pulp tissue in 24h under the conditions of 2-4 DEG C.
Dental pulp is extracted
The pre-treatment of Isolated Tooth：First, the tooth body 2 times of Isolated Tooth, each 1min are rinsed using tooth storing liquid；Then,
Utilize 1% Povidone Iodine Disinfection Solution (PVP-I) immersion Isolated Tooth 1min；Finally, then with tooth storing liquid Isolated Tooth is rinsed 2 times,
Each 1min.
Dental pulp is extracted：First, dentistry annular jumping through rings incisor body neck portion (not Exposed Pulp) is utilized；Then, nothing is utilized
Bacterium gauze wrapped tooth body, tooth body is separated, Exposed Pulp, is placed in the culture dish containing tooth storing liquid；Finally, spy is utilized
The medicine equipments such as pin, tweezers separate dental pulp, are placed in sterile PBS, as shown in Figure 1.
Pulp tissue digests and stem cell culture
The pulp tissue of extraction is placed in sterile PBS, using micro- eye scissors, shredding pulp tissue, (size is about
1mm2)；Then, pulp tissue is collected using centrifuge tube, and in 1000rpm under normal temperature condition, centrifuges 10min.Supernatant is abandoned, is added dropwise
3mg/mL type i collagen enzyme+4mg/mL neutral proteinases mixed enzymes (volume ratio is 1: 1) dispel pulp tissue, are placed in 37 DEG C,
Digested in 180rpm constant-temperature table environment.Digestion is terminated respectively at blood serum medium behind 5,10,15 and 20min, is added dropwise,
It is collected by centrifugation tissue block and cell (1000rpm, 5min), the complete α-MEM culture mediums of 1mL (20%FBS+100U/mL penicillin+
100ug/mL streptomysins) it is resuspended, it is inoculated in 35-mm sterile petri dish, is put into 37 DEG C, 5%CO2Carried out in cell culture incubator
Fluid infusion 2mL after culture, 1d, a subculture is changed every 3d.Wherein, pulp tissue's half digestion in 37 DEG C of isoperibols
30min and totally disappeared 60min (cell suspension utilizes the filtering of 70um cell sieves) as a control group, other condition of culture and experimental group
Unanimously, experimental result is as shown in Figure 2.
The passage of dental pulp stem cell
When cell fusion reaches 80%-90%, digested with pancreatin/EDTA mixed enzymes, centrifugation (1000rpm,
5min), cell is collected, with complete α-MEM culture medium cell dispersions, cell suspension is evenly distributed in 2 Tissue Culture Dish, put
In 37 DEG C, 5%CO2Cultivated in incubator, liquid is changed once every 3d.
The biological function detection of dental pulp stem cell
CCK-8 is detected：Choose the preferable P of growth conditions1For dental pulp stem cell, adjustment cell concentration is 2.0 × 103Inoculation
In 96- porocyte culture plates, recovery nutrient solution is added, is put into 37 DEG C, 5%C02 incubators and is cultivated, changed every 3d
One subculture.Respectively at after culture 1,3 and 5d, 10 μ L CCK-8 solution is added per hole, is put into cell culture incubator and continues to train
1h is supported, the absorbance (0D values) at 450nm is then observed on ELIASA, as a result as shown in figure 3, TES is 10min, dental pulp
The proliferation activity of stem cell apparently higher than other experimental groups and control group, have obvious significant difference (*P ＜ 0.05versus TE
30min,#P ＜ 0.05versus E 60min).
Immunofluorescence dyeing：10min P will be digested under the conditions of constant-temperature table3Six orifice plates are inoculated in for dental pulp stem cell to climb
On piece, after culture terminates, 4% paraformaldehyde fixes 30min, BSA joint 0.1TritonX-100 rupture of membranes closings 30min；
4 DEG C of overnight incubations of CD146, STRO-1 primary antibody；PBST is rinsed 3 times, and secondary antibody lucifuge is incubated 1h, and PBST is rinsed 3 times, and DAPI lucifuges are incubated
5min is educated, PBST is rinsed 3 times；Fluorescence microscope is made film.
Into fat to differentiation：Choose the P of exponential phase3For dental pulp stem cell (digesting 10min under the conditions of constant-temperature table), often
Rule digestion, the cell suspension of gained is according to 20000/cm2Density be inoculated in six orifice plates, complete α-MEM culture mediums are added dropwise,
It is placed in 37 DEG C, 5%CO2Cultivated in incubator, liquid is changed once every 3d, when cell fusion degree reaches 100% or so,
OriCell is added in complete α-MEM cell culture mediumsTMAdipogenic induction agent (Cyagen, USA), is put into CO2Enter in cell culture incubator
Row culture, the cell culture medium once containing adipogenic induction agent is changed every 3d.Cell is continuously cultivated after 21d, terminates induction,
4% paraformaldehyde (PFA) fixes 10min, oil red-O staining reagents, observation by light microscope.
Skeletonization is to differentiation：Choose the P of exponential phase3For dental pulp stem cell (digesting 10min under the conditions of constant-temperature table), often
Rule digestion, the cell suspension of gained is according to 20000/cm2Density be inoculated in 35-mm Tissue Culture Dish, be added dropwise complete
α-MEM culture mediums, are placed in 37 DEG C, 5%CO2Cultivated in incubator, when cell fusion degree reaches 70% or so, complete
OriCell is added in α-MEM cell culture mediumsTMOsteogenic induction agent (Cyagen, USA), is put into CO2Trained in cell culture incubator
Support, the cell culture medium once containing osteogenic induction agent is changed every 3d.Cell is continuously cultivated after 21d, terminates induction, room temperature bar
Under part, 4% paraformaldehyde (PFA) fixes 10min, alizarin red-S staining reagent 3min, observation by light microscope.
Into cartilage to differentiation：Choose the P of exponential phase3For dental pulp stem cell (digesting 10min under the conditions of constant-temperature table),
Conventional digestion, adjustment concentration of cell suspension is 5 × 105Individual/mL, takes 1mL cell suspensions to be put into 15mL centrifuge tubes, normal temperature centrifugation
(1000rpm, 5min) collects cell, abandons supernatant, is directly added into the centrifuge tube containing OriCellTMInto chondrocyte induction agent
Complete α-MEM the cell culture mediums (adition process must be slow, in order to avoid break up cell) of (Cyagen, USA), cell is with cell mass
Form be put into 37 DEG C, 5%CO2Cultivated in incubator, liquid is changed once every 3d.After 28d, induction, 4% poly first are terminated
Aldehyde (PFA) fixation is stayed overnight, specimens paraffin embedding slices (4-5 μm), alcian blue staining reagent, observation by light microscope.
Dental pulp stem cell freezes and recovered
Dental pulp stem cell freezes：The dental pulp stem cell for meeting standard logarithmic growth period is taken, when cell fusion reaches 80%-
When 90%, digested, centrifuged (1000rpm, 5min) with pancreatin/EDTA mixed enzymes, collected cell, use cells frozen storing liquid
(10%DMSO+90%FBS) adjustment cell concentration is 3 × 106Individual/mL, cryopreservation tube is respectively charged into by cell suspension 1mL
In (1.5mL), cryopreservation tube number consecutively is then put into the special freezing storing box (temperature drop rate of cell：1 DEG C/min) in, put
Enter -80 DEG C of refrigerators and freeze 12h, last cell cryopreservation tube is transferred in liquid nitrogen to be stored for a long time, and carries out corresponding registration.
The recovery of dental pulp stem cell：The principle of rapid fluid resuscitation is carried out, after cell cryopreservation tube takes out from liquid nitrogen, is quickly put into
Thawed, be then diluted with 5mL complete α-MEM culture mediums in 37 DEG C of water baths, centrifuged (1000rpm, 5min),
Cell is collected, is dispelled with complete α-MEM culture mediums, is transferred in 35-mm steril cell culture dishes, is put into 37 DEG C, 5%CO2Cell
Cultivated in incubator, change a subculture within 3 days.
Described above is only the preferred embodiment of the present invention, and protection scope of the present invention is not limited merely to above-mentioned implementation
Example, all technical schemes belonged under thinking of the present invention belong to protection scope of the present invention.It should be pointed out that for the art
Those of ordinary skill for, some improvements and modifications without departing from the principles of the present invention, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (8)
1. a kind of preparation method of primary dental pulp stem cell, it is characterised in that comprise the following steps：
(1) tooth body is collected；
(2) dental pulp is extracted：Dental pulp is extracted after the Isolated Tooth of collection is carried out into pre-treatment；
(3) pulp tissue's digestion and stem cell culture：The pulp tissue of extraction is placed in sterile PBS, pulp tissue is shredded extremely
1mm2, then, pulp tissue is collected using centrifuge tube, and in 1000rpm under normal temperature condition, centrifuge 10min, abandon supernatant, it is added dropwise
3mg/mL type i collagen enzyme+4mg/mL neutral proteinase mixed enzymes dispel pulp tissue, type i collagen enzyme and neutral proteinase volume
Ratio is 1: 1, is placed in 37 DEG C, 180rpm constant-temperature table environment and is digested, after 5-10min, and blood serum medium is added dropwise and terminates
Under the conditions of digestion, 1000rpm, centrifugal treating 5min collects tissue block and cell, and the complete α-MEM culture mediums of 1mL are resuspended, are inoculated in
In 35mm sterile petri dish, in 37 DEG C, 5%CO2Cultivated in cell culture incubator, complete α-MEM culture medium fluid infusion after 1d
2mL, a α-MEM culture medium is changed every 3d；
(4) passage processing is carried out to dental pulp stem cell.
2. a kind of preparation method of primary dental pulp stem cell according to claim 1, it is characterised in that described complete α-
MEM culture mediums are 10-20%FBS+100U/mL penicillin+100ug/mL streptomysins.
3. a kind of preparation method of primary dental pulp stem cell according to claim 1, it is characterised in that described step
(2) pre-treatment is the tooth body 2 times that Isolated Tooth is rinsed using tooth storing liquid during dental pulp is extracted, and then each 1min gathers using 1%
Ketone iodic disinfecting liquid bubble Isolated Tooth 1min is tieed up, finally Isolated Tooth is rinsed 2 times with tooth storing liquid again, each 1min.
4. a kind of preparation method of primary dental pulp stem cell according to claim 1, it is characterised in that described step
(4) passage process step is carried out to dental pulp stem cell is：It is mixed with pancreatin/EDTA when pulp cells fusion reaches 80%-90%
Synthase is digested, and 5min is centrifuged under the conditions of 1000rpm, collects cell, and with complete α-MEM culture medium cell dispersions, cell hangs
Liquid is evenly distributed in 2 Tissue Culture Dish, is placed in 37 DEG C, 5%CO2Cultivated in incubator, liquid is changed once every 3d.
5. a kind of method for building dental pulp stem cell storehouse, it is characterised in that comprise the following steps：
(1) tooth body is collected；
(2) tooth body is transported；
(3) dental pulp is extracted；
(4) pulp tissue's digestion and stem cell culture；
(5) passage of dental pulp stem cell；
(6) the biological function detection of dental pulp stem cell；
(7) dental pulp stem cell freezes and recovered.
6. a kind of method for building dental pulp stem cell storehouse according to claim 5, it is characterised in that described dental pulp is dry thin
The biological function detection of born of the same parents includes CCK-8 detections, immunofluorescence dyeing detection, examined into fat to differentiation detection, skeletonization to differentiation
Survey or the one or more into cartilage into differentiation detection.
7. a kind of method for building dental pulp stem cell storehouse according to claim 5, it is characterised in that described step (7)
Freezing for dental pulp stem cell comprises the following steps：The dental pulp stem cell for meeting standard logarithmic growth period is taken, when cell fusion reaches
During 80%-90%, digested with pancreatin/EDTA mixed enzymes, 5min is centrifuged under the conditions of 1000rpm, cell is collected, with 10%
DMSO+90%FBS cells frozen storing liquid, adjustment cell concentration is 3 × 106Individual/mL, cell suspension 1mL is respectively charged into and frozen
Cryopreservation tube number consecutively, is then put into the special freezing storing box of cell by Guan Zhong, is cooled with 1 DEG C/min speed, is put into -80 DEG C of refrigerators
12h is frozen, last cell cryopreservation tube is transferred in liquid nitrogen to be stored for a long time, and carries out corresponding registration.
8. a kind of method for building dental pulp stem cell storehouse according to claim 5, it is characterised in that described step (7)
The recovery of dental pulp stem cell comprises the following steps：After cell cryopreservation tube takes out from liquid nitrogen, quickly it is put into 37 DEG C of water baths
Thawed, be then diluted with 5mL complete α-MEM culture mediums, 5min is centrifuged under the conditions of 1000rpm, collect cell, used
Complete α-MEM culture mediums are dispelled, and are transferred in 35-mm steril cell culture dishes, are put into 37 DEG C, 5%CO2Carried out in cell culture incubator
Culture, changes a subculture in 3 days.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108517312A (en) * | 2018-04-20 | 2018-09-11 | 重庆医科大学附属口腔医院 | A kind of preparation method of the outer assessment models of lid marrow material bodies |
| CN109321522A (en) * | 2018-11-20 | 2019-02-12 | 潍坊市康华生物技术有限公司 | A method of preparing dental pulp stem cell |
| CN109628389A (en) * | 2018-12-24 | 2019-04-16 | 北京博奥晶典启衡生物科技有限公司 | Culture for amplification in vitro dental pulp stem cell freezes system, method and kit |
| CN110229787A (en) * | 2019-06-17 | 2019-09-13 | 华夏源（上海）细胞基因工程股份有限公司 | A kind of primary extracting method of dental pulp stem cell |
| CN112251401A (en) * | 2020-10-16 | 2021-01-22 | 浙江优牙生物科技有限公司 | Large-scale normal-temperature digestion method for dental pulp stem cells |
| CN112458048A (en) * | 2020-12-09 | 2021-03-09 | 浙江优牙生物科技有限公司 | Adherence promoting matrix for primary culture of dental pulp stem cells and culture method of primary dental pulp stem cells |
| CN112980783A (en) * | 2021-03-18 | 2021-06-18 | 优牙生物科技(上海)有限公司 | Method for improving secretion capacity of dental pulp stem cells |
| CN114651811A (en) * | 2022-02-22 | 2022-06-24 | 张亮 | Additive for storing dental pulp mesenchymal stem cells and application thereof |
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| CN109321522A (en) * | 2018-11-20 | 2019-02-12 | 潍坊市康华生物技术有限公司 | A method of preparing dental pulp stem cell |
| CN109628389A (en) * | 2018-12-24 | 2019-04-16 | 北京博奥晶典启衡生物科技有限公司 | Culture for amplification in vitro dental pulp stem cell freezes system, method and kit |
| CN110229787A (en) * | 2019-06-17 | 2019-09-13 | 华夏源（上海）细胞基因工程股份有限公司 | A kind of primary extracting method of dental pulp stem cell |
| CN110229787B (en) * | 2019-06-17 | 2022-10-14 | 华夏源细胞工程集团股份有限公司 | Primary extraction method of dental pulp stem cells |
| CN112251401A (en) * | 2020-10-16 | 2021-01-22 | 浙江优牙生物科技有限公司 | Large-scale normal-temperature digestion method for dental pulp stem cells |
| CN112458048A (en) * | 2020-12-09 | 2021-03-09 | 浙江优牙生物科技有限公司 | Adherence promoting matrix for primary culture of dental pulp stem cells and culture method of primary dental pulp stem cells |
| CN112980783A (en) * | 2021-03-18 | 2021-06-18 | 优牙生物科技(上海)有限公司 | Method for improving secretion capacity of dental pulp stem cells |
| CN114651811A (en) * | 2022-02-22 | 2022-06-24 | 张亮 | Additive for storing dental pulp mesenchymal stem cells and application thereof |
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Address after: 325000 East of Science and Technology Business Incubator Innovation Building, Venture Service Center, Wenzhou High-tech Industrial Development Zone, Zhejiang Province Applicant after: Zhejiang excellent tooth Biotechnology Co., Ltd. Applicant after: WENZHOU BIOMEDICAL MATERIALS AND ENGINEERING RESEARCH INSTITUTE Applicant after: Wenzhou Medical University Address before: 325000 Technology Business Incubator R&D Headquarters Building, Venture Service Center, Wenzhou High-tech Industrial Development Zone, Zhejiang Province, 14 floors Applicant before: Wenzhou excellent Biotechnology Co., Ltd. Applicant before: WENZHOU BIOMEDICAL MATERIALS AND ENGINEERING RESEARCH INSTITUTE Applicant before: Wenzhou Medical University |
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Application publication date: 20171020 |