CN107114357A - The frozen stock solution and its cryopreservation methods of a kind of periodontal ligament stem cell - Google Patents
The frozen stock solution and its cryopreservation methods of a kind of periodontal ligament stem cell Download PDFInfo
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- CN107114357A CN107114357A CN201710442567.8A CN201710442567A CN107114357A CN 107114357 A CN107114357 A CN 107114357A CN 201710442567 A CN201710442567 A CN 201710442567A CN 107114357 A CN107114357 A CN 107114357A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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Abstract
The invention belongs to the frozen stock solution and its cryopreservation methods in cell cryopreservation field, more particularly to a kind of periodontal ligament stem cell.The frozen stock solution of the present invention includes glycerine, mesenchymal stem cell serum-free nutrient solution and human serum albumin.It is poor that the invention can effectively solve existing frozen stock solution using effect, the low technological deficiency of periodontal ligament stem cell motility rate after recovery.The periodontal ligament stem cell frozen stock solution of the present invention can not only keep freeze-stored cell activity, and periodontal ligament stem cell motility rate is significantly improved after recovery, not influence expression and the differentiation potential of stem cell of Stem cell surface marker thing also.
Description
Technical field
The invention belongs to the frozen stock solution and its cryopreservation methods in cell cryopreservation field, more particularly to a kind of periodontal ligament stem cell.
Background technology
Periodontal disease is not only the main cause for causing adult to lose tooth, or harm Human Oral Cavity or even whole body health
Main oral disease.Periodontosis is the infectious diseases under the chronic inflammation stimulation of periodontium, is the microorganism in bacterial plaque
Inflammation caused by under corresponding microenvironment, it damages the destruction of periodontium, especially alveolar bone and the forfeiture of periodontium,
Ultimately result in tooth mobility come off and alveolar bone absorption.And the target of periodontal treatment seeks to treat periodontal disease, improve
Microenvironment regenerates new connective tissue and adhered to and supporting tissue again, completes the structure of alveolar bone, cementum and parodontium.Often
Periodontal regenerative method has guided tissue regeneration, root planing etc., and such method is simple and easy to apply but can not be completely real
, there is corresponding limitation in existing feature and structural paradenlal tissue regeneration.The appearance of periodontal tissue engineering technology and develop into
Periodontal regenerative provides brand-new idea and method.
Parodontium is one layer of connective tissue between root of the tooth and alveolar bone, is important Periodontal Supporting Tissue, mainly by into
Fibrocyte, cementoblast and some undifferentiated mesenchymal cell compositions.Under normal circumstances, periodontium has itself
Renewal and repair ability, its repair action realized by periodontal ligament cell self-renewing.In disease or by the external world
During stimulation, regeneration is made by the continuous propagation and differentiation of cell in parodontium.Seo in 2004 etc. is from the third molar pulled out
Isolated periodontal ligament stem cell (Periodontal Ligament Sem Cells, PDLSCs) in periodontal membrane tissue.In addition,
The inner surface of tooth socket is also with the presence of PDLSCs after extraction.Recently, someone has obtained highly purified from human adult periodontal tissue
PDLSCs clone.It is worth noting that, more researchs are found, also separated from inflammatory periodontal membrane tissue and obtain PDLSCs,
And its potential for still keeping regenerating cementum and periodontal membrane tissue is proved, also the not enough disadvantage in its source is being compensate for a certain degree
End.Research shows that PDLSCs expresses the label STRO-1 and CD146/MUC18 of mescenchymal stem cell, it means that PDLSCs
Probably it is derived from circumvascular cell mass.And this point is exactly with same expression STRO-1's and CD146/MUC18
Mesenchymal stem cells MSCs (Bone Marrow Mesenchymal Stem Cells, BMMSCs) is closely similar.PDLSCs
With the Multidirectional Differentiation ability similar to BMSCs, can vitro differentiation be various kinds of cell, such as fat cell, cartilage cell, nerve are thin
Born of the same parents etc., important function has been played in the stable state of periodontium, the metabolic processes for safeguarding alveolar bone are maintained.
Although there is research to carry out periodontal again using the adult stem cell in the Various Tissues source including fat, marrow
It is raw, but its development is still constrained the problems such as the invasive and moral check of materials.Therefore, PDLSCs is expected to turn into periodontium again
Raw preferred seed cell.
In recent years, stem cell bank set up the need for and the clinically increase in demand to stem cell, to stem cell cryopreserving with
Research in terms of recovery causes increasing concern.As the MSCs of other sources, PDLSCs is ground as organizational engineering
Study carefully the seed cell with potential clinical practice, its excessively passage can show obvious aging or apoptosis, and long-term in vitro culture is easily sent out
Raw Spontaneous Differentiation, loses many differentiation potentials, and propagation, Adhering capacity decline, apoptosis rate increase, so freezing PDLSCs and being also
It studies one of important step of application.
The solution used when cells frozen storing liquid is a kind of cell cryopreservation, it seeks necessary to can supplying cell life metabolism
Material is supported, while damaging action of the freezing ice crystal to cell can be prevented or reduced.The cells frozen storing liquid or commercially available commonly used at present
Cells frozen storing liquid in be typically by dimethyl sulfoxide (DMSO) (dimathyl sulfoxide, DMSO) and hyclone (fetal
Bovine serum, FBS) mixing composition.DMSO is that a kind of molecular weight is small, and the chemistry with strong solvability and penetrating power
Material.In many researchs, DMSO is the most frequently used cell cryopreservation protective agent, with its preserve cell recovery after with fresh cells
Compare, with similar phenotype, cell surface marker and growth rate.DMSO mechanism of action is through thin in temperature-fall period
After birth enters intracellular, electrolyte concentration in the reduction inside and outside solution that do not freeze of cell, so as to protect cells from high concentration electrolysis
Matter is damaged, while ICW will not excessively exosmose, it is to avoid cell transition is dehydrated shrinkage.However, DMSO has necessarily to cell
Toxic action, excessive concentration can cause higher osmotic pressure, be unfavorable for cell recovery.Therefore, the conventional concentrations of current DMSO
For 5%-15%.FBS belongs to heterologous material, complicated component, and there is the risk for introducing pollution and anaphylactogen, is not suitable for facing
Bed application.Particularly in cell therapy, the presence of heterologous protein, it is unnecessary to cause, even it is unknown not
Good reaction, has a strong impact on treatment results.
In summary, traditional cells frozen storing liquid can produce toxicity and immunogenic response to periodontal ligament stem cell, therefore,
Research and development are a kind of not to produce cytotoxicity, immunogenicity to periodontal ligament stem cell, moreover it is possible to significantly improve recovery motility rate after cell cryopreservation
Frozen stock solution be those skilled in the art's technical problem urgently to be resolved hurrily.
The content of the invention
In view of this, the invention provides a kind of frozen stock solution of periodontal ligament stem cell and its cryopreservation methods, can effectively it solve
Traditional frozen stock solution produces poor, the Cell viability after recovery that freezes effect caused by toxicity and immunogenic response to periodontal ligament stem cell
Low technological deficiency.
A kind of frozen stock solution for periodontal ligament stem cell that the present invention is provided, including glycerine, mesenchymal stem cell serum-free culture
Liquid and human serum albumin.
Preferably, the frozen stock solution includes：The volumn concentration of glycerine is that 10%, mesenchymal stem cell serum-free is trained
The volumn concentration of nutrient solution is that 30-60%, the volumn concentration of human serum albumin are 30-60%.
Preferably, the frozen stock solution includes：The volumn concentration of glycerine is that 10%, mesenchymal stem cell serum-free is trained
The volumn concentration of nutrient solution is that the volumn concentration of 40%, human serum albumin is 50%.
Preferably, the human serum albumin mass concentration is 20%.
Further, the invention also discloses a kind of periodontal ligament stem cell cryopreservation methods, comprise the following steps：To parodontium
Frozen after the frozen stock solution, mixing are added in stem cell.
Preferably, the addition of frozen stock solution be to periodontal ligament stem cell density be 1.0 × 106Individual/mL-5 × 106Individual/
mL。
Preferably, described freeze after specially described frozen stock solution is mixed with the periodontal ligament stem cell is dispensed into cryopreservation tube
It is interior, it is put into after the program temperature reduction box for recovering room temperature in advance, places -80 DEG C of ultra low temperature freezers, Liquid nitrogen storage is transferred to after 2-3 days.
The present invention discloses a kind of periodontal ligament stem cell frozen stock solution, including glycerine, mesenchymal stem cell serum-free nutrient solution and
Human serum albumin.Pass through the synergy of glycerine, mesenchymal stem cell serum-free nutrient solution and human serum albumin three.Wherein,
Glycerine can penetrate into intracellular, outer production in the cell as permeability cryoprotector, before the solidification completely of cell freezing suspension
Raw certain molar concentration, reduction intraor extracellular does not freeze the concentration of electrolyte in solution, so as to protect cells from high concentration
The damage of electrolyte, while ICW also will not too exosmose, it is to avoid cell is too dehydrated shrinkage；Mescenchymal stem cell
Serum-free medium and human serum albumin can maintain cell normal osmotic pressure, keep cell membrane integrity, maintain normal pH value with
And protection cell membrane surface protein function is normal.The present invention has been greatly improved permeability of the cell membrane to water, can make intracellular
Moisture emigrated cell outside, the formation of intracellular ice crystal is reduced, so as to reduce due to the cellular damage that causes of ice crystal formation.This freezes
Liquid storage is free of DMSO, it is to avoid its toxic action to cell, while do not contain animal sources serum, it is to avoid introduce pollution and mistake
Quick former risk, has higher clinical safety compared with regular growth frozen stock solution.Meanwhile, periodontal ligament stem cell of the invention
Frozen stock solution and cryopreservation methods can keep freezing the activity of periodontal ligament stem cell, and periodontal ligament stem cell motility rate is significantly improved after recovery,
And the expression of cell surface marker thing is not influenceed, its differentiation potential is had no effect on, lipoblast and skeletonization is particularly divided into
The potential of cell.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the accompanying drawing used required in technology description to be briefly described.
Fig. 1 shows periodontal ligament stem cell surface marker expression rate figure；
Fig. 2 shows cell recovery result and the Cell viability statistics using embodiment recovery cell；
Fig. 3 shows the cell growth curve figure using comparative example culture；
Fig. 4 shows the cell growth curve cultivated using embodiment 3；
Fig. 5 show use comparative example and embodiment 3 to cultivate cell growth morphological image (left-hand column for comparative example, right hand column
It is first, second and third day cell growth figure for embodiment the 3, the 1st, 2,3 rows)；
Fig. 6 show use comparative example and embodiment 3 to cultivate cell growth morphological image (left-hand column for comparative example, right hand column
Be fourth, fifth for embodiment the 3, the 1st, 2,3 rows, six days cell growth figures)；
Fig. 7 show use comparative example and embodiment 3 to cultivate cell growth morphological image (left-hand column for comparative example, right hand column
It is the 7th day cell growth figure for the row of embodiment the 3, the 1st)；
Fig. 8 shows the PDLSCs surface marker expression rate figures of embodiment 3；
Fig. 9 shows comparative example and the Osteoblast Differentiation design sketch (200 ×) of embodiment 3；
Figure 10 shows comparative example and embodiment 3 into fat differentiation effect figure (200 ×).
Wherein, CD146-A, CD105-A, CD73-A, HLA-DR-A, CD34-A, CD45-A in Fig. 1 and Fig. 8 abscissas
For surface antigen CD146, CD105, CD73, HLA-DR, CD34, CD45, ordinate is antigen presentation rate.
Embodiment
The invention provides a kind of periodontal ligament stem cell frozen stock solution and cryopreservation methods, traditional frozen stock solution can be effectively solved to tooth
All film stem cells, which produces, freezes that effect is poor caused by toxicity and immunogenic response, the low technological deficiency of Cell viability after recovery.
The technical scheme in the embodiment of the present invention will be clearly and completely described below, it is clear that described implementation
Example only a part of embodiment of the invention, rather than whole embodiments.Based on the embodiment in the present invention, this area is common
The every other embodiment that technical staff is obtained under the premise of creative work is not made, belongs to the model that the present invention is protected
Enclose.
Wherein, present invention mesenchymal stem cell serum-free nutrient solution used is commercially available prod：LONZA12-725F,
UltraCULTURE Serum-free Medium(SFM)。
Embodiment 1
It is primary to PDLSCs progress to be separately cultured, identify and pass on.
PDLSCs progress is primary to be separately cultured, and is comprised the following steps：
1st, draw materials：Take 12-18 Sui health donors because correction needs the pull out the 3rd to grind one's teeth in sleep, quick immersion 4 DEG C of precoolings
In dual anti-PBS containing 3 times of volumes.
2nd, rinse：Tooth body is rinsed with the direction containing 3 times of dual anti-PBS from root of the tooth to corona, blood stains is thoroughly removed, repeats 3
It is secondary.
3rd, periodontium is scraped：Lower 1/3 section in root of periodontal membrane tissue is unidirectionally scraped with knife blade crown root, centrifugation is moved into
Pipe, and tissue block is cut into about 1mm with eye scissors3Size.
4th, digest：Add appropriate clostridiopetidase A-Dispase enzymes 1:1 mixture slaking liquid (clostridiopetidase A 3g/L, Dispase enzyme 4g/
L), put in 37 DEG C of constant-temperature tables and digest 30-35min.After digestion terminates, 1000r/min centrifugation 5min abandon supernatant.Add appropriate
PBS is resuspended, 1000r/min centrifugation 5min, abandons supernatant.
5th, it is inoculated with：Add nutrient solution (the DMEM nutrient solutions of the 10%FBS containing volume fraction) and cell is resuspended, be inoculated in six holes
Plate, 5%CO237 DEG C of cultures of incubator.
6th, purify：After after the 80%-90% that cell covers with cell plates area, cell is collected, is purified with magnetic bead sorting method
Cell, by the cell of collection and STRO-1 monoclonal antibodies after 4 DEG C are incubated 1h, 4 DEG C of incubation 30min is mixed with magnetic bead, in magnetic force devices
STRO-1 and PDLSCs are filtered out under effect, PDLSCs is inoculated in six new orifice plates, in 5%CO237 DEG C of cultures of incubator.
PDLSCs is identified, comprised the following steps：After cell covers with the 80-90% of platen area, cell is collected
(about 1 × 106It is individual), add after 3g/L Triton immersions 20min, PBS is washed 3 times, 10% lowlenthal serum room temperature closing 2h, respectively
It is added dropwise 1:Each 50 μ L of CD146, CD105, CD73, HLA-DR, CD34, CD45 antibody of 200 dilutions, while control group (is not incubated
Negative group of antibody) pure PBS50 μ L are added dropwise, 4 DEG C of processing 16h, next day room temperature places 30min, and PBS is rinsed 3 times, and each group is dripped respectively
Plus 1:The anti-igg of FITC marks two of 500 dilutions, normal temperature lucifuge is incubated 1h, and PBS is rinsed 2 times, and 10% formalin fixes cell,
Flow cytometer determines the positive rate of antigen.
As a result show, compared with control group, PDLSCs surface antigens CD146, CD105, CD73 expression of separation are positive, and
HLA-DR, CD34, CD45 expression are negative, illustrate to be successfully separated from tooth to PDLSCs.
The PDLSCs cell surface marker expression rates of table 1
| Cell phenotype | CD146 | CD105 | CD73 | HLA-DR | CD34 | CD45 |
| Positive expression rate (%) | 99.80 | 99.80 | 99.80 | 0.20 | 0.20 | 0.30 |
PDLSCs is passed on, and is comprised the following steps：Result to be identified is positive PDLSCs bed boards, covers with platen area
After 80-90%, inhaled with suction pipe and abandon old nutrient solution, add 0.25% trypsase, digested 1-3 minutes, Microscopic observation cellular contraction
Become bowlder, the DMEM nutrient solutions containing 10% percent by volume FBS are added immediately and terminate digestion, collect cell, 1000r/min centrifugations
5min, abandons supernatant.The DMEM nutrient solutions containing 10% percent by volume FBS are added, cell count are carried out, by 5 × 104Individual/mL is close
Degree, which is inoculated in culture dish, carries out Secondary Culture, 5%CO237 DEG C of cultures of incubator, liquid was changed every 2-3 days once.
Comparative example
The preparation of comparative example frozen stock solution：By frozen stock solution volumn concentration for 10% DMSO, frozen stock solution volume basis contains
The frozen stock solution that comparative example is made after the FBS mixing for 90% is measured, it is standby in 4 DEG C of refrigerator cold-storages.
Embodiment 2
The preparation of the frozen stock solution of embodiment 2：Glycerine, embodiment 2 by the volumn concentration of the frozen stock solution of embodiment 2 for 10%
The volumn concentration of frozen stock solution is 30% mesenchymal stem cell serum-free nutrient solution and the volume basis of the frozen stock solution of embodiment 2
The human serum albumin (human serum albumin mass concentration is 20%) that content is 60%, is made the frozen stock solution of embodiment 2 after mixing, in
4 DEG C of refrigerator cold-storages are standby.
Embodiment 3
The preparation of the frozen stock solution of embodiment 3：By the volumn concentration of the frozen stock solution of embodiment 3 for 10% glycerine, will implement
Mesenchymal stem cell serum-free nutrient solution of the volumn concentration of the frozen stock solution of example 3 for 40% and the body by the frozen stock solution of embodiment 3
The human serum albumin (human serum albumin mass concentration is 20%) that product percentage composition is 50%, is made the jelly of embodiment 3 after mixing
Liquid storage, it is standby in 4 DEG C of refrigerator cold-storages.
Embodiment 4
The preparation of the frozen stock solution of embodiment 4：The glycerine for being 10% by volumn concentration, volumn concentration are between 50%
(human serum albumin mass concentration is for 40% human serum albumin for mesenchymal stem cells serum-free medium and volumn concentration
20%) frozen stock solution of embodiment 4, is made after mixing, it is standby in 4 DEG C of refrigerator cold-storages.
Embodiment 5
The preparation of the frozen stock solution of embodiment 5：By the glycerine by the volumn concentration of the frozen stock solution of embodiment 3 for 10%, general's reality
Apply the volumn concentration of the frozen stock solution of example 3 for 60% mesenchymal stem cell serum-free nutrient solution and by the frozen stock solution of embodiment 3
The human serum albumin (human serum albumin mass concentration is 20%) that volumn concentration is 30%, is made embodiment 5 after mixing
Frozen stock solution, it is standby in 4 DEG C of refrigerator cold-storages.
Embodiment 6
Cell cryopreservation step is：
1st, cell is collected：The 3rd generation PDLSCs is chosen, treats that cell covers with the 80-90% of flat board, is inhaled with suction pipe and abandons old culture
Liquid, adds appropriate 0.25% trypsase, digests 1-3 minutes, and Microscopic observation cellular contraction becomes bowlder, is filled between adding in right amount immediately
Matter stem cell serum-free culture medium terminates digestion, collects cell, and 1000r/min centrifugation 5min abandon supernatant.
2nd, freeze：Respectively with each embodiment and comparative example frozen stock solution freeze-stored cell prepared, frozen stock solution addition is extremely
Periodontal ligament stem cell density is 1.0 × 106Individual/mL, setting is repeated 3 times.It is dispensed into 2mL cryopreservation tubes, 1.5mL/ pipes are put into and carried
The preceding program temperature reduction box for recovering room temperature, places -80 DEG C of ultra low temperature freezers, and program temperature reduction box can guarantee that temperature with 1 DEG C/min speed
Degree declines；Liquid nitrogen storage is transferred to after 2 days.Embodiment 7
The cell of each embodiment and comparative example is recovered, comprised the following steps：What comparative example froze with each embodiment
Cell repeats cell recovery in after liquid nitrogen cryopreservation one month, taking out three groups respectively, and cryopreservation tube takes out and is immediately placed in 37 DEG C of water
Dissolved in bath, need constantly to rock cryopreservation tube in course of dissolution.After 1-2min liquid melts, with 10mL mescenchymal stem cells without blood
Clear culture medium diluting cells suspension (cryopreservation tube is washed one time with culture medium), takes 0.5mL cell suspensions to carry out cytometer after mixing
Number and Activity determination.Experimental result shown, PDLSCs frozen stock solutions of the present invention, which freeze effect and are substantially better than regular growth, to be frozen
Liquid (table 2, Fig. 2).
Table 2 is counted using the cell recovery result and Cell viability of embodiment recovery cell
Embodiment 8
The cell growth curve of embodiment 3 and comparative example is compared, comprised the following steps：Comparative example freezes with embodiment 3
Cell in after liquid nitrogen cryopreservation one month, cryopreservation tube, which takes out to be immediately placed in 37 DEG C of water-baths, to be dissolved, and is needed in course of dissolution constantly
Rock cryopreservation tube.After 1-2min liquid melts, with 10mL mesenchymal stem cell serum-free culture medium diluting cells suspension (with culture
Base is washed cryopreservation tube one time), 1000r/min centrifugation 5min abandon supernatant.With the mescenchymal stem cell of the EGF containing 10ng/ml without blood
After clear culture medium is resuspended, cell presses 1 × 104Individual/mL density is inoculated in 24 orifice plates, is put into 5%CO237 DEG C of cultures of incubator.Often
It is collected cell and carries out cell count, and each random collecting calculates 3 repetitions (hole 1, hole 2, hole 3), continuous 7 days, draws cell
Growth curve.As a result show and PDLSCs is frozen using people PDLSCs frozen stock solutions of the present invention, cell growth rule are cultivated after recovery
Rule is normal, and its proliferation activity freezes PDLSCs higher than conventional cells frozen storing liquid, as shown in table 3 and table 4, Fig. 3, Fig. 4.It is thin daily
Born of the same parents collect the preceding growthform of two groups of cells of Continuous Observation under inverted microscope and gather image.As a result as shown in Figure 5-Figure 7.
Fig. 3-Fig. 7 results illustrate that the frozen stock solution of the embodiment of the present invention 3 promotes proliferation function to having after cell recovery.
The daily count results of MDSCs recovery cultures that the comparative example of table 3 freezes
The daily count results of MDSCs recovery cultures that the embodiment 3 of table 4 freezes
Embodiment 9
The cell surface marker thing of embodiment 3 is determined, comprised the following steps：The cell that embodiment 3 freezes freezes in liquid nitrogen
Deposit after one month, cryopreservation tube, which takes out to be immediately placed in 37 DEG C of water-baths, to be dissolved, and needs constantly to rock cryopreservation tube in course of dissolution.1-
After 2min liquid melts, (cryopreservation tube is washed with culture medium with 10mL mesenchymal stem cell serum-free culture medium diluting cells suspensions
One time), 1000r/min centrifugation 5min abandon supernatant.With 10ml EGF containing 10ng/ml mesenchymal stem cell serum-free culture medium
After resuspension, cell is inoculated in 10cm culture dishes, is put into 5%CO237 DEG C of cultures of incubator.Cell, fluidic cell are collected after 48h
Instrument detects its surface marker CD146, CD105, CD73, HLA-DR, CD34 and CD45 expression.As a result such as table 5 and figure
8。
Testing result shows, using periodontal ligament stem cell frozen stock solution freeze-stored cell of the present invention, the PDLSCs after recovery
Express the typical surface marker of stem cell, with freeze before PDLSCs compared that there was no significant difference.Show of the present invention
PDLSCs frozen stock solutions do not interfere with the expression of PDLSCs cell surface marker thing.
The embodiment 3PDLSCs cell surface marker expression rates of table 5
Embodiment 10
Multi-lineage potential is detected, is comprised the following steps：The cell that comparative example and embodiment 3 freeze is in liquid nitrogen cryopreservation one
After month, cryopreservation tube, which takes out to be immediately placed in 37 DEG C of water-baths, to be dissolved, and needs constantly to rock cryopreservation tube in course of dissolution.1-2min liquid
After thawing, (cryopreservation tube is washed one time with culture medium) with 10mL mesenchymal stem cell serum-free culture medium diluting cells suspension,
1000r/min centrifuges 5min, abandons supernatant.After being resuspended with the mesenchymal stem cell serum-free culture medium of the EGF containing 10ng/mL, cell
By 1 × 105Individual/mL density is inoculated in 6 orifice plates, is put into 5%CO237 DEG C of cultures of incubator.Treat cell fusion degree up to 80% with
On, the periodontal ligament stem cell after normal periodontal ligament stem cell and the cryopreservation resuscitation of embodiment 3 that position froze carries out induced osteogenesis point
Change and break up into fat.Oil red O stain is carried out to two composition fat Analytical Chemical Experiment group cells after 14 days, to two groups of Osteoblast Differentiations after 21 days
Experimental group cell carries out Alizarin red staining.As shown in figure 9, the negative control and adipogenic induction group of comparative example and the moon of embodiment 3
Property control it is similar with adipogenic induction group coloration result, Figure 10 show comparative example negative control and osteogenic induction group and embodiment 3
Negative control it is similar with osteogenic induction group coloration result, should test result indicates that periodontal ligament stem cell frozen stock solution of the present invention
Do not interfere with PDLSCs into fat and Osteoblast Differentiation potential (Fig. 9, Figure 10).
In summary, the invention can effectively solve existing frozen stock solution to freeze effect to periodontal ligament stem cell poor, after recovery
The low technological deficiency of Cell viability.The periodontal ligament stem cell frozen stock solution of the present invention can keep freeze-stored cell activity, recovery very well again
Periodontal ligament stem cell motility rate is significantly improved afterwards, and does not influence the expression of cell surface marker thing and its into the differentiation of fat and skeletonization
Potential.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (7)
1. a kind of frozen stock solution of periodontal ligament stem cell, it is characterised in that including glycerine, mesenchymal stem cell serum-free nutrient solution and
Human serum albumin.
2. the frozen stock solution of periodontal ligament stem cell according to claim 1, it is characterised in that the frozen stock solution includes：Glycerine
Volumn concentration be that the 10%, volumn concentration of mesenchymal stem cell serum-free nutrient solution is 30-60%, the white egg of human blood
White volumn concentration is 30-60%.
3. the frozen stock solution of periodontal ligament stem cell according to claim 2, it is characterised in that the frozen stock solution includes：Glycerine
Volumn concentration be that the 10%, volumn concentration of mesenchymal stem cell serum-free nutrient solution is 40%, human serum albumin
Volumn concentration be 50%.
4. the frozen stock solution of periodontal ligament stem cell according to claim 3, it is characterised in that the human serum albumin quality is dense
Spend for 20%.
5. a kind of periodontal ligament stem cell cryopreservation methods, add described in Claims 1-4 any one into periodontal ligament stem cell and freeze
Frozen after liquid storage, mixing.
6. cryopreservation methods according to claim 5, it is characterised in that the addition of frozen stock solution is to periodontal ligament stem cell density
For 1.0 × 106Individual/mL-5 × 106Individual/mL.
7. cryopreservation methods according to claim 6, it is characterised in that described to freeze specially described frozen stock solution and the periodontal
Film stem cell is dispensed into cryopreservation tube after mixing, and is put into after the program temperature reduction box for recovering room temperature in advance, is placed -80 DEG C of ultralow temperature ices
Case, is transferred to Liquid nitrogen storage after 2-3 days.
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| CN109430252A (en) * | 2018-12-25 | 2019-03-08 | 成都赋智健康科技有限公司 | A kind of stem cell cryopreserving liquid and preparation method thereof |
| CN109601527A (en) * | 2018-12-27 | 2019-04-12 | 广州赛莱拉干细胞科技股份有限公司 | A kind of periodontal ligament stem cell frozen stock solution and its cryopreservation methods |
| CN111418580A (en) * | 2020-05-25 | 2020-07-17 | 山东万能干细胞生物技术有限公司 | Stem cell cryopreservation solution and cryopreservation method |
| CN112715533A (en) * | 2021-02-26 | 2021-04-30 | 康妍葆(北京)干细胞科技有限公司 | Cryopreservation solution and cryopreservation method for mesenchymal stem cells |
| CN112772639A (en) * | 2021-03-25 | 2021-05-11 | 徐会娟 | Periodontal ligament stem cell cryopreservation liquid and cryopreservation method |
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