CN105861429A - Resuscitation culture medium and resuscitation culture method for dental pulp mesenchymal stem cells - Google Patents
Resuscitation culture medium and resuscitation culture method for dental pulp mesenchymal stem cells Download PDFInfo
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- CN105861429A CN105861429A CN201610235181.5A CN201610235181A CN105861429A CN 105861429 A CN105861429 A CN 105861429A CN 201610235181 A CN201610235181 A CN 201610235181A CN 105861429 A CN105861429 A CN 105861429A
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- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Abstract
The invention relates to the technical field of cell culture, in particular to a resuscitation culture medium and a resuscitation culture method for dental pulp mesenchymal stem cells. A preparation method for the resuscitation culture medium comprises the steps that the dental pulp mesenchymal stem cells are taken, subjected to first culture through a complete culture medium and then subjected to second culture through a serum-free medium, liquid supernatant is collected, and the resuscitation culture medium is obtained. The resuscitation culture medium for the dental pulp mesenchymal stem cells is used for carrying out resuscitation culture on the dental pulp mesenchymal stem cells, the resuscitation vigor of the dental pulp mesenchymal stem cells can be improved easily, the cells can be proliferated, and the biological characteristics of the dental pulp mesenchymal stem cells can be kept better.
Description
Technical field
The present invention relates to technical field of cell culture, particularly to the recovery of a kind of dental pulp mescenchymal stem cell
Culture medium and recovery cultural method thereof.
Background technology
Periodontitis involves four kinds of Periodontal Supporting Tissue (gum, parodontium, alveolar bone and cementum)
Chronic infectious disease, often causes the inflammatory destruction of Periodontal Supporting Tissue.After 35 years old the most common.
Common disease is because of bacterial plaque, dental calculus, traumatogenic articulation, food impaction, ill fitting prosthesis, mouth breathing etc..
Periodontitis has four big features, i.e. oral pocket formation, the inflammation of bag wall, absorption of alveolar bone, tooth are gradually
Loosen.Serious periodontitis can cause loss of tooth, thus cause masticatory function low and cause digestion not
Good and gastrointestinal disease, affects physical and mental health.
Essentially consisting in of periodontitis for the treatment of diminishes inflammation, and promotes the regeneration of destroyed periodontium, recovers
The normal function of tooth.Clinical frequently with periodontitis treatment method include: periodental non-surgical treatment (scaling,
Scrape control, root planing), periodontal flap surgery and paradenlal tissue regeneration art.
Periodental non-surgical treatment: scaling, also referred to as cleans one's teeth, and is commonly called as toothwash, and it is prevention and the one for the treatment of odontopathy
The method of kind.I.e. remove the pigment of attachment on supragingival calculus, bacterial plaque and facing with scaling apparatus, and polish tooth
Face, is to prevent bacterial plaque and dental calculus redeposited, prevents and treats the measure of periodontosis.Also should be by gingival sulcus when scaling
The interior a part of subgingival calculus being connected with supragingival calculus is disposed the most in the lump.It is different according to apparatus used,
Supragingival scaling is divided into hand instrument scaling method and ultrasonic cavitron scaling method.For gingivitis patients,
Every 6～12 months make a scaling, can effectively safeguard periodontal health.Subgingival curettage, i.e. root planing
Art, its be subgingival debridement apparatus by hand stretch into remove in oral pocket be attached in oral pocket on root face and
Embed the subgingival calculus in cementum and bacterial plaque, and strike off the root surface pathology dentale by endotoxin contamination
Matter, thus form smooth, the root face of cleaning, make root mask have biocompatible surface, beneficially periodontal
The attachment of tissue and new life.Root planing shall not be applied to healthy periodontal loci, in order to avoid causing periodontal to adhere to
Lose.
Periodontal flap surgery: refer to use different operative incisions, separated with the tissue of lower section by gum, is formed
Gingiva tissue lobe, exposes root face and the alveolar bone of diseased region, it is provided that debridement approach and visuality.Strike off disease
After becoming tissue and bacterial plaque dental calculus, gingival flap is resetted and goes up in place and sew up, reach to eliminate periodontal
Bag or make the purpose that oral pocket shoals.
Paradenlal tissue regeneration art: at the alveolar bone district implantable artificial bone lacked, and cover special life
Thing guiding film, grows on root face, thus reaches parodontium, tooth with making the cell selective on parodontium
Groove bone and cemental regeneration.Planted and biomembranous technology by this bone, recover the alveolus destroyed
Bone, gum and other periodontium, complete the again stable of tooth.
Above method cannot repair periodontal attachment and the alveolus tissue of damage, it is impossible to it is the most right to meet
Oral and maxillofacial defect reparative regeneration and function, the requirement of profile recovery, secondly periodontitis treatment leads to
Cross antibiosis and usually control bacterial plaque growth, carry out whole body and the drug therapy of local.But antibiotic side effect is relatively
Many, pathogenic microorganism also can produce drug resistance.
Dental pulp stem cell (dental pulp stem cells, DPSCs) is present in tooth pulp tissue
One class has self, multiplication capacity by force, and the mescenchymal stem cell of Multidirectional Differentiation ability.Dental pulp is done
Cell has the potential of Multidirectional Differentiation, and it is except forming the extracellular of Mineral nodules ability, through difference
The induction of cell factor, additionally it is possible to be divided into fat, bone, cartilage, muscle, blood vessel endothelium, liver, god
Through etc. clone type.It is reported, dental pulp stem cell can play in periodontitis treatment and well repair work
With, mechanism of action is: by implanting local and being divided into defect cell, secrete cytokines, chemotactic is done
Cell, to local, suppresses local inflammation, promotes that local vascular is newborn.
Frozen, the resuscitation technique of dental pulp stem cell, cell is related in the clinical practice of dental pulp stem cell
Recovery is by long-term Cryopreservation cell in liquid nitrogen, thaws to normal temperature process rapidly, guarantees simultaneously
Recover activity and the biological property of cell.At present, the complete medium pair of general commercial is generally used
Dental pulp stem cell carries out recovery and cultivates.But the complete medium of general commercial is in the resuscitation process of cell
Cannot ensure the vigor of stem cell, cell proliferation rate is relatively low, affects the clinical utilization of stem cell.Therefore,
Need to develop a kind of recovery medium that can preferably maintain dental pulp mescenchymal stem cell vigor and recovery thereof
Method.
Summary of the invention
In view of this, the invention provides recovery medium and the recovery thereof of a kind of dental pulp mescenchymal stem cell
Cultural method.This recovery medium can preferably maintain the vigor of dental pulp mescenchymal stem cell.
In order to realize foregoing invention purpose, the present invention provides techniques below scheme:
The invention provides the recovery medium of a kind of dental pulp mescenchymal stem cell, the system of this recovery medium
Preparation Method is: take dental pulp mescenchymal stem cell, after using complete medium to carry out the first cultivation, then uses
After serum free medium carries out the second cultivation, collect supernatant, it is thus achieved that recovery medium.
As preferably, the first time cultivated was 24～72h.
In some embodiments that the present invention provides, the first time cultivated was 24h.
In some embodiments that the present invention provides, first cultivates as at 37 DEG C, 5%CO2Under the conditions of cultivate.
As preferably, the inoculum density of the first dental pulp mescenchymal stem cell cultivated be (5～10) ×
103cell/cm2。
Preferably, the inoculum density of the first dental pulp mescenchymal stem cell cultivated is 7 × 103cell/cm2。
As preferably, serum free medium is DMEM/F12 culture medium.
As preferably, the second time cultivated was 24～72h.
In some embodiments that the present invention provides, the second time cultivated was 24h, 48h or 72h.
In some embodiments that the present invention provides, second cultivates as at 37 DEG C, 5%CO2Under the conditions of cultivate.
As preferably, after collecting supernatant, also include the step being centrifuged.
As preferably, being centrifuged is 1000～2000rpm to be centrifuged 5～10min.
In some embodiments that the present invention provides, it is centrifuged and is centrifuged 5min for 1000rpm.
As preferably, after being centrifuged, also include the step of filtration sterilization.
In some embodiments that the present invention provides, it is 0.22 μm for the aperture of the filter membrane of filtration sterilization.
Present invention also offers the recovery cultural method of a kind of dental pulp mescenchymal stem cell, use the present invention's
Frozen dental pulp mescenchymal stem cell is cultivated in recovery medium recovery.
As preferably, the recovery cultural method of the dental pulp mescenchymal stem cell that the present invention provides is: take frozen
Dental pulp mescenchymal stem cell, after incubation, use recovery medium of the present invention recovery to cultivate after incubation
Dental pulp mescenchymal stem cell.
Preferably, the recovery cultural method of the dental pulp mescenchymal stem cell that the present invention provides is: take frozen
Dental pulp mescenchymal stem cell, shakes incubation 30～60s at 35～42 DEG C, adds the present invention of 5～10 times of volumes
Recovery medium, centrifugal, remove supernatant, by (5～10) × 103cell/cm2Density to be inoculated in the present invention multiple
Soviet Union's culture medium, at 37 DEG C, 5%CO2Under the conditions of cultivate 24h, it is thus achieved that the first recovery dental pulp mescenchymal stem cell；
Then by (5～10) × 103cell/cm2Density the first recovery dental pulp mescenchymal stem cell is inoculated in this
Bright recovery medium, at 37 DEG C, 5%CO2Under the conditions of continue cultivate 24～48h, it is thus achieved that the dental pulp after recovery
Mescenchymal stem cell.
In some embodiments that the present invention provides, the recovery of the dental pulp mescenchymal stem cell that the present invention provides
Cultural method is: take frozen dental pulp mescenchymal stem cell, shakes incubation 60s, add 5 times of bodies at 42 DEG C
Long-pending recovery medium of the present invention, centrifugal, remove supernatant, by 7 × 103cell/cm2Density be inoculated in this
Bright recovery medium, at 37 DEG C, 5%CO2Under the conditions of cultivate 24h, it is thus achieved that the first recovery dental pulp mesenchyma do
Cell；Then by 7 × 103cell/cm2Density the first recovery dental pulp mescenchymal stem cell is inoculated in this
Bright recovery medium, at 37 DEG C, 5%CO2Under the conditions of continue cultivate 48h, it is thus achieved that fill between the dental pulp after recovery
Matter stem cell.
The invention provides recovery medium and the recovery cultural method thereof of a kind of dental pulp mescenchymal stem cell.
The preparation method of this recovery medium is: take dental pulp mescenchymal stem cell, uses complete medium to carry out the
After one cultivates, then after using serum free medium to carry out the second cultivation, collect supernatant, it is thus achieved that recovery training
Support base.The invention have the benefit that and carry out between dental pulp with the conditioned medium of dental pulp mescenchymal stem cell
The recovery of mesenchymal stem cells is cultivated, and beneficially dental pulp mescenchymal stem cell brings back to life raising and the cell increasing of vigor
Grow, it is possible to preferably keep the biological property of dental pulp mescenchymal stem cell.
Accompanying drawing explanation
Fig. 1 shows cell viability testing result after embodiment 1 recovery；
Fig. 2 shows the flow cytometer detection result of the dental pulp mescenchymal stem cell in embodiment 1 after the recovery of test group；
Wherein Fig. 2-1 shows that CD73 testing result, Fig. 2-2 show that CD90 testing result, Fig. 2-3 show CD105 testing result,
Fig. 2-4 shows that CD34 testing result, Fig. 2-5 show that CD45 testing result, Fig. 2-6 show HLA-DR testing result；
Fig. 3 shows the flow cytometer detection result of the dental pulp mescenchymal stem cell in embodiment 1 after the recovery of control group；
Wherein Fig. 3-1 shows that CD73 testing result, Fig. 3-2 show that CD90 testing result, Fig. 3-3 show CD105 testing result,
Fig. 3-4 shows that CD34 testing result, Fig. 3-5 show that CD45 testing result, Fig. 3-6 show HLA-DR testing result；
Fig. 4 shows the induced osteogenesis differentiation mirror of the dental pulp mescenchymal stem cell in embodiment 1 after the recovery of test group
Determine result；
Fig. 5 shows that the induction of the dental pulp mescenchymal stem cell in embodiment 1 after the recovery of test group becomes fat differentiation mirror
Determine result；
Fig. 6 shows cell viability testing result after embodiment 2 recovery；
Fig. 7 shows the flow cytometer detection result of the dental pulp mescenchymal stem cell in embodiment 2 after the recovery of test group；
Wherein Fig. 7-1 shows that CD73 testing result, Fig. 7-2 show that CD90 testing result, Fig. 7-3 show CD105 testing result,
Fig. 7-4 shows that CD34 testing result, Fig. 7-5 show that CD45 testing result, Fig. 7-6 show HLA-DR testing result；
Fig. 8 shows the flow cytometer detection result of the dental pulp mescenchymal stem cell in embodiment 2 after the recovery of control group；
Wherein Fig. 8-1 shows that CD73 testing result, Fig. 8-2 show that CD90 testing result, Fig. 8-3 show CD105 testing result,
Fig. 8-4 shows that CD34 testing result, Fig. 8-5 show that CD45 testing result, Fig. 8-6 show HLA-DR testing result；
Fig. 9 shows cell viability testing result after embodiment 3 recovery；
Figure 10 shows the flow cytometer detection result of the dental pulp mescenchymal stem cell in embodiment 3 after the recovery of test group；
Wherein Figure 10-1 shows that CD73 testing result, Figure 10-2 show that CD90 testing result, Figure 10-3 show that CD105 detects
As a result, Figure 10-4 shows that CD34 testing result, Figure 10-5 show that CD45 testing result, Figure 10-6 show HLA-DR
Testing result；
Figure 11 shows the flow cytometer detection result of the dental pulp mescenchymal stem cell in embodiment 3 after the recovery of control group；
Wherein Figure 11-1 shows that CD73 testing result, Figure 11-2 show that CD90 testing result, Figure 11-3 show that CD105 detects
As a result, Figure 11-4 shows that CD34 testing result, Figure 11-5 show that CD45 testing result, Figure 11-6 show HLA-DR
Testing result.
Detailed description of the invention
The invention discloses recovery medium and the recovery cultural method thereof of a kind of dental pulp mescenchymal stem cell,
Those skilled in the art can use for reference present disclosure, is suitably modified technological parameter and realizes.Of particular note
, all similar replacements and change apparent to those skilled in the art, they are all
It is deemed to be included in the present invention.Method and the application of the present invention are described by preferred embodiment,
Related personnel substantially can in without departing from present invention, spirit and scope to method described herein and should
With being modified or suitably changing and combine, realize and apply the technology of the present invention.
Term is explained:
Cell recovery: by long-term Cryopreservation cell in liquid nitrogen, thaws rapidly to normal temperature process,
Guarantee to recover activity and the biological property of cell simultaneously；
Conditioned medium: in cell proliferation incubation, in cell meeting secretion activity material to nutrient solution,
Wherein cell factor is one of main active material, this cultivated certain cell or tissue after, contain
The nutrient solution having cell secreta is just called conditioned medium.Utilize conditioned medium can be effectively improved cell
Or the success rate of tissue in vitro culture, and different cells, tissue are had by the conditioned medium of separate sources
Different promotions or inhibitory action.
Used by the recovery medium of the dental pulp mescenchymal stem cell that the present invention provides and recovery cultural method thereof
Biomaterial all can be buied by market.
Below in conjunction with embodiment, the present invention it is expanded on further:
Embodiment 1
(1) cultivation of dental pulp mescenchymal stem cell and the collection of conditioned medium
(1) cultivation of dental pulp mescenchymal stem cell
Collect the healthy tooth coming off with servant and extracting for 30 years old, PBS dental surface dirt, use clamp
Schizodont tooth, Exposed Pulp tissue；Pulp tissue is gripped with aseptic nipper.Shred, place 50mL centrifuge tube
In, add the 3g/L type i collagen enzyme of 10 times of volumes, be sufficiently mixed after sealing uniformly, be transferred to Tempeerature-constant air
In shaking table, 37 DEG C, 200R digest about 10-20min, add isopyknic complete medium terminate digestion,
Piping and druming discrete cellular agglomerate repeatedly, by the cell screen filtration of 70 μm, to obtain single discrete cell,
1000rpm is centrifuged 5min.Abandoning supernatant, precipitate 1 time with 30mL PBS, 1000rpm is centrifuged 5min.
Precipitation DMEM/F12+10%FBS is cultivated resuspended, with 0.5～1 × 104/cm2It is seeded in six orifice plates, often
Hole adds 2mL growth medium, adds EGF EGF and Basic Fibroblast Growth Factor
BFGF (both final concentration of 10ng/mL), mix cell suspension, be put in 37 DEG C, humidity be the two of 95%
Carbonoxide incubator is cultivated.Every 3d changes liquid 1 time, and about 7-14d is under the microscope it is observed that the shape of clone
Become, when cell grow to 80%-90% converge time, 0.125% trypsin digestion and cell, carry out passing on training
Support.By the resuspended precipitation of complete medium after Li Xin, 7 × 103/cm2Cell density inoculated and cultured ware continues training
Support.Take P3-P5 and carry out conditioned medium collection for cell.
(2) conditioned medium is collected
For the dental pulp mescenchymal stem cell of conditioned medium, first according to 7 × 103/cm2Cell density inoculation training
Support ware, after complete medium carries out cultivating 24 hours, after PBS, add the DMEM/F12 of serum-free
After culture medium continues to cultivate 24 hours, collect cell culture medium supernatant, supernatant is sub-packed in 50mL centrifuge tube
In, 1000pm is centrifuged 5min.Remove precipitation, collect supernatant.CMC model is drawn with disposable syringe
Base, with 0.22 μm membrane filtration, is collected in sterile culture flask.It is placed on-20 DEG C of incubators to preserve 6 months
About-12 months or be placed on-80 DEG C preserve more than 1 year.When needing to use, return to normal temperature and use.
(2) recovery of dental pulp mescenchymal stem cell
Recovery test is divided into test group and control group, and the operating process of test group is: by between frozen dental pulp
Frozen mescenchymal stem cell is taken out from liquid nitrogen by mesenchymal stem cells, is placed in the water-bath of 42 DEG C, shake
Swing incubation 60 seconds, after cell thaws, immediately frozen stock solution is joined the above-mentioned condition collected of 5 times of volumes
In culture medium, take a small amount of cell suspension, add trypan blue, be placed on cell viability calculating instrument and carry out cell
Viability examination, remaining cell suspension is centrifuged 5 minutes with 1000rpm, removes supernatant, resuspended with conditioned medium
Precipitation；Blow and beat mixing gently, and according to 7 × 103/cm2In cell density inoculated and cultured ware, use CMC model
Base is cultivated.It is placed on 37 DEG C, 5%CO2After incubator stands 24 hours, change conditioned medium and continue training
Supporting, observation of cell amplification every day situation, cell carries out continuing to cultivate 48 hours, centrifugal thin with trypsinization
Born of the same parents, use conditioned medium re-suspended cell, take a small amount of cell suspension, add trypan blue, are placed on cell and live
Power calculating instrument carries out cell viability detection, and remaining cell carries out passage or other experiments.
The operating process of control group is: takes out dental pulp mescenchymal stem cell from liquid nitrogen, puts into 42 DEG C of water immediately
Bath is melted.After PBS washing, take out cell and carry out cell viability detection, be centrifuged 5 minutes with 1000rpm,
Remove supernatant, use complete medium re-suspended cell, according to 7 × 103/cm2In cell density inoculated and cultured ware, and
It is positioned over 37 DEG C, 5%CO2Incubator is cultivated 72 hours.Use trypsinization centrifuge cell, use CMC model
Base re-suspended cell, takes a small amount of cell suspension, adds trypan blue, is placed on cell viability calculating instrument and carries out carefully
Born of the same parents' viability examination.Remaining cell carries out passage or other experiments.Viability examination result is shown in Fig. 1.
As shown in Figure 1, test group cell brings back to life vigor and is higher than control group, shows the present embodiment recovery medium
Dental pulp mescenchymal stem cell is conducive to bring back to life raising and the cell proliferation of vigor, it is possible to preferably to keep dental pulp
The biological property of mescenchymal stem cell.
(3) the stem cell surface mark after flow cytomery recovery
After recovering, cell confluency degree reaches 80%-90%, with 0.25% trypsinization centrifuge cell, and counts
After often pipe add 2 × 105Cell number, dye solution washes 1 time, and 1000rpm is centrifuged 5min；Abandon supernatant, use
Dye solution piping and druming mixing cell；Add CD73, CD90, CD105, CD34, CD45 and
The each 2 μ L of HLA-DRA antibody, and set a pipe as blank；At 4 DEG C, lucifuge reaction 15-20min；
Dye solution is washed once, and 1000rpm is centrifuged 5min；Directly the cell of mark abandons supernatant, and lucifuge adds
The sample-loading buffer of 500 μ L, mixing, with 200 eye mesh screen filtration cell samples, flow cytomery is thin
Cellular surface antigen.The result of the test of test group is shown in Fig. 2, and the result of the test of control group is shown in Fig. 3.
From Fig. 2, Fig. 3, test group uses the recovery medium of present invention offer to frozen mesenchyma
After stem cell carries out recovery cultivation, negative surface marker CD34, CD45, HLA-DR are all to present feminine gender,
Mescenchymal stem cell surface marker CD73, CD90 simultaneously, CD105 all present the positive.After recovery is described
Dental pulp mescenchymal stem cell still keeps the biological property of mescenchymal stem cell.
(4) after recovery, cell induction Osteoblast Differentiation is identified
After to be tested group of recovery, cell confluency degree reaches 80%-90%, with 0.25% trypsinization centrifuge cell,
According to 5 × 103Cell/cm2It is inoculated in six orifice plates, after adding complete medium cultivation 24h, adds skeletonization and lure
Leading culture medium and carry out Fiber differentiation, changed liquid every 2-3 days, carry out Alizarin red staining after 28 days, result is shown in Fig. 4.
From fig. 4, it can be seen that the mescenchymal stem cell after Fu Su, after osteogenic induction 4 weeks, alizarin red contaminate
Calcium scoring seen from look, illustrates the dental pulp mescenchymal stem cell after recovery medium of the present invention is recovered still
So keep the potential to Osteoblast Differentiation.
(5) after recovery, mescenchymal stem cell induces into fat differentiation qualification
After to be tested group of recovery, cell confluency degree reaches 80%-90%, with 0.25% trypsinization centrifuge cell,
After collecting cell, and according to 5 × 103Cell/cm2It is inoculated in six orifice plates, after adding complete medium 24h,
Add adipogenic induction culture medium to cultivate.Adipogenic induction culture medium include basal medium DMEM, 10%
Hyclone, 0.5mM IBMX, 1 μM of dexamethasone, 100 μMs of Indomethacins, 5 μ g/mL pancreas islet
Element, 2mm/L glutamine etc..Within every three days, change liquid.Carry out oil red O stain after surrounding, identify that fat drips formation
Situation, result is shown in Fig. 5.
As seen from Figure 5, the mescenchymal stem cell after recovery, after adipogenic induction 4 weeks, by oil red O stain
Red color visible oil droplet, illustrates the dental pulp mescenchymal stem cell after recovery medium of the present invention is recovered still
Keep the potential to Adipose Differentiation.
Embodiment 2
(1) cultivation of dental pulp mescenchymal stem cell and the collection of conditioned medium
(1) cultivation of dental pulp mescenchymal stem cell
Collect the healthy tooth coming off with servant and extracting for 30 years old, PBS dental surface dirt, use clamp
Schizodont tooth, Exposed Pulp tissue；Pulp tissue is gripped with aseptic nipper.Shred, place 50mL centrifuge tube
In, add the 3g/L type i collagen enzyme of 10 times of volumes, be sufficiently mixed after sealing uniformly, be transferred to Tempeerature-constant air
In shaking table, 37 DEG C, 200R digest about 10-20min, add isopyknic complete medium terminate digestion,
Piping and druming discrete cellular agglomerate repeatedly, by the cell screen filtration of 70 μm, to obtain single discrete cell,
1000rpm is centrifuged 5min.Abandoning supernatant, precipitate 1 time with 30mL PBS, 1000rpm is centrifuged 5min.
Precipitation DMEM/F12+10%FBS is cultivated resuspended, with 0.5～1 × 104/cm2It is seeded in six orifice plates, often
Hole adds 2mL growth medium, adds EGF EGF and Basic Fibroblast Growth Factor
BFGF (both final concentration of 10ng/mL), mix cell suspension, be put in 37 DEG C, humidity be the two of 95%
Carbonoxide incubator is cultivated.Every 3d changes liquid 1 time, and about 7-14d is under the microscope it is observed that the shape of clone
Become, when cell grow to 80%-90% converge time, 0.125% trypsin digestion and cell, carry out passing on training
Support.By the resuspended precipitation of complete medium after Li Xin, 7 × 103/cm2Cell density inoculated and cultured ware continues training
Support.Take P3-P5 and carry out conditioned medium collection for cell.
(2) conditioned medium is collected
For the dental pulp mescenchymal stem cell of conditioned medium, first according to 7 × 103/cm2Cell density inoculation training
Support ware, after complete medium carries out cultivating 24 hours, after PBS, add the DMEM/F12 of serum-free
After culture medium continues to cultivate 48 hours, collect cell culture medium supernatant, supernatant is sub-packed in 50mL centrifuge tube
In, 1000rpm is centrifuged 5min.Remove precipitation, collect supernatant.CMC model is drawn with disposable syringe
Base, with 0.22 μm membrane filtration, is collected in sterile culture flask.It is placed on-20 DEG C of incubators to preserve 6 months
About-12 months or be placed on-80 DEG C preserve more than 1 year.When needing to use, return to normal temperature and use.
(2) recovery of dental pulp mescenchymal stem cell
Recovery test is divided into test group and control group, and the operating process of test group is: by between frozen dental pulp
Frozen mescenchymal stem cell is taken out from liquid nitrogen by mesenchymal stem cells, is placed in the water-bath of 42 DEG C, shake
Swing incubation 60 seconds, after cell thaws, immediately frozen stock solution is joined the above-mentioned condition collected of 5 times of volumes
In culture medium, take a small amount of cell suspension, add trypan blue, be placed on cell viability calculating instrument and carry out cell
Viability examination, remaining cell suspension is centrifuged 5 minutes with 1000rpm, removes supernatant, resuspended with conditioned medium
Precipitation；Blow and beat mixing gently, and according to 7 × 103/cm2In cell density inoculated and cultured ware, use CMC model
Base is cultivated.It is placed on 37 DEG C, 5%CO2After incubator stands 24 hours, change conditioned medium and continue training
Supporting, observation of cell amplification every day situation, cell carries out continuing to cultivate 48 hours, centrifugal thin with trypsinization
Born of the same parents, use conditioned medium re-suspended cell, take a small amount of cell suspension, add trypan blue, are placed on cell and live
Power calculating instrument carries out cell viability detection, and remaining cell carries out passage or other experiments.
The operating process of control group is: takes out dental pulp mescenchymal stem cell from liquid nitrogen, puts into 42 DEG C of water immediately
Bath is melted.After PBS washing, take out cell and carry out cell viability detection, be centrifuged 5 minutes with 1000rpm,
Remove supernatant, use complete medium re-suspended cell, according to 7 × 103/cm2In cell density inoculated and cultured ware, and
It is positioned over 37 DEG C, 5%CO2Incubator is cultivated 72 hours.Use trypsinization centrifuge cell, use CMC model
Base re-suspended cell, takes a small amount of cell suspension, adds trypan blue, is placed on cell viability calculating instrument and carries out carefully
Born of the same parents' viability examination.Remaining cell carries out passage or other experiments.Cell viability testing result is shown in Fig. 6.
It will be appreciated from fig. 6 that test group cell brings back to life vigor is higher than control group, show the present embodiment recovery medium
Dental pulp mescenchymal stem cell is conducive to bring back to life raising and the cell proliferation of vigor, it is possible to preferably to keep dental pulp
The biological property of mescenchymal stem cell.
(3) the stem cell surface mark after flow cytomery recovery
After recovering, cell confluency degree reaches 80%-90%, with 0.25% trypsinization centrifuge cell, and counts
After often pipe add 2 × 105Cell number, dye solution washes 1 time, and 1000rpm is centrifuged 5min；Abandon supernatant, use
Dye solution piping and druming mixing cell；Add CD73, CD90, CD105, CD34, CD45 and
The each 2 μ L of HLA-DRA antibody, and set a pipe as blank；At 4 DEG C, lucifuge reaction 15-20min；
Dye solution is washed once, and 1000rpm is centrifuged 5min；Directly the cell of mark abandons supernatant, and lucifuge adds
The sample-loading buffer of 500 μ L, mixing, with 200 eye mesh screen filtration cell samples, flow cytomery is thin
Cellular surface antigen.The result of the test of test group is shown in Fig. 7, and the result of the test of control group is shown in Fig. 8.
From Fig. 7, Fig. 8, test group uses the recovery medium of present invention offer to frozen mesenchyma
After stem cell carries out recovery cultivation, negative surface marker CD34, CD45, HLA-DR are all to present feminine gender,
Mescenchymal stem cell surface marker CD73, CD90 simultaneously, CD105 all present the positive.After recovery is described
Dental pulp mescenchymal stem cell still keeps the biological property of mescenchymal stem cell.
(4) after recovery, cell induction Osteoblast Differentiation is identified
After to be tested group of recovery, cell confluency degree reaches 80%-90%, with 0.25% trypsinization centrifuge cell,
According to 5 × 103Cell/cm2It is inoculated in six orifice plates, after adding complete medium cultivation 24h, adds skeletonization and lure
Lead culture medium and carry out Fiber differentiation, changed liquid every 2-3 days, after 28 days, carry out Alizarin red staining, result and Fig. 4
Similar.Visible, after recovery mescenchymal stem cell, after osteogenic induction 4 weeks, can by Alizarin red staining
See calcium scoring, illustrate that the dental pulp mescenchymal stem cell after recovery medium of the present invention is recovered still is protected
Hold the potential to Osteoblast Differentiation.
(5) after recovery, mescenchymal stem cell induces into fat differentiation qualification
After to be tested group of recovery, cell confluency degree reaches 80%-90%, with 0.25% trypsinization centrifuge cell,
After collecting cell, and according to 5 × 103Cell/cm2It is inoculated in six orifice plates, after adding complete medium 24h,
Add adipogenic induction culture medium to cultivate.Adipogenic induction culture medium include basal medium DMEM, 10%
Hyclone, 0.5mM IBMX, 1 μM of dexamethasone, 100 μMs of Indomethacins, 5 μ g/mL pancreas islet
Element, 2mm/L glutamine etc..Within every three days, change liquid.Carry out oil red O stain after surrounding, identify that fat drips formation
Situation, result is similar to Fig. 5.Visible, after recovery mescenchymal stem cell, after adipogenic induction 4 weeks,
By oil red O stain red color visible oil droplet, the dental pulp mesenchyma after recovery medium of the present invention is recovered is described
Stem cell remains in that the potential to Adipose Differentiation.
Embodiment 3
(1) cultivation of dental pulp mescenchymal stem cell and the collection of conditioned medium
(1) cultivation of dental pulp mescenchymal stem cell
Collect the healthy tooth coming off with servant and extracting for 30 years old, PBS dental surface dirt, use clamp
Schizodont tooth, Exposed Pulp tissue；Pulp tissue is gripped with aseptic nipper.Shred, place 50mL centrifuge tube
In, add the 3g/L type i collagen enzyme of 10 times of volumes, be sufficiently mixed after sealing uniformly, be transferred to Tempeerature-constant air
In shaking table, 37 DEG C, 200R digest about 10-20min, add isopyknic complete medium terminate digestion,
Piping and druming discrete cellular agglomerate repeatedly, by the cell screen filtration of 70 μm, to obtain single discrete cell,
1000rpm is centrifuged 5min.Abandoning supernatant, precipitate 1 time with 30mL PBS, 1000rpm is centrifuged 5min.
Precipitation DMEM/F12+10%FBS is cultivated resuspended, with 0.5～1 × 104/cm2It is seeded in six orifice plates, often
Hole adds 2mL growth medium, adds EGF EGF and Basic Fibroblast Growth Factor
BFGF (both final concentration of 10ng/mL), mix cell suspension, be put in 37 DEG C, humidity be the two of 95%
Carbonoxide incubator is cultivated.Every 3d changes liquid 1 time, and about 7-14d is under the microscope it is observed that the shape of clone
Become, when cell grow to 80%-90% converge time, 0.125% trypsin digestion and cell, carry out passing on training
Support.By the resuspended precipitation of complete medium after Li Xin, 7 × 103/cm2Cell density inoculated and cultured ware continues training
Support.Take P3-P5 and carry out conditioned medium collection for cell.
(2) conditioned medium is collected
For the dental pulp mescenchymal stem cell of conditioned medium, first according to 7 × 103/cm2Cell density inoculation training
Support ware, after complete medium carries out cultivating 24 hours, after PBS, add the DMEM/F12 of serum-free
Culture medium, after continuing to cultivate 72 hours, collects cell culture medium supernatant, supernatant is sub-packed in 50mL and is centrifuged
Guan Zhong, 1000pm are centrifuged 5min.Remove precipitation, collect supernatant.Condition training is drawn with disposable syringe
Support base, with 0.22 μm membrane filtration, be collected in sterile culture flask.It is placed on-20 DEG C of incubators and preserves 6
Individual month about-12 months or be placed on-80 DEG C preserve more than 1 year.When needing to use, return to normal temperature and use.
(2) recovery of dental pulp mescenchymal stem cell
Recovery test is divided into test group and control group, and the operating process of test group is: by between frozen dental pulp
Frozen mescenchymal stem cell is taken out from liquid nitrogen by mesenchymal stem cells, is placed in the water-bath of 42 DEG C, shake
Swing incubation 60 seconds, after cell thaws, immediately frozen stock solution is joined the above-mentioned condition collected of 5 times of volumes
In culture medium, take a small amount of cell suspension, add trypan blue, be placed on cell viability calculating instrument and carry out cell
Viability examination, remaining cell suspension is centrifuged 5 minutes with 1000rpm, removes supernatant, resuspended with conditioned medium
Precipitation；Blow and beat mixing gently, and according to 7 × 103/cm2In cell density inoculated and cultured ware, use CMC model
Base is cultivated.It is placed on 37 DEG C, 5%CO2After incubator stands 24 hours, change conditioned medium and continue training
Supporting, observation of cell amplification every day situation, cell carries out continuing to cultivate 48 hours, centrifugal thin with trypsinization
Born of the same parents, use conditioned medium re-suspended cell, take a small amount of cell suspension, add trypan blue, are placed on cell and live
Power calculating instrument carries out cell viability detection, and remaining cell carries out passage or other experiments.
The operating process of control group is: takes out dental pulp mescenchymal stem cell from liquid nitrogen, puts into 42 DEG C of water immediately
Bath is melted.After PBS washing, take out cell and carry out cell viability detection, be centrifuged 5 minutes with 1000rpm,
Remove supernatant, use complete medium re-suspended cell, according to 7 × 103/cm2In cell density inoculated and cultured ware, and
It is positioned over 37 DEG C, 5%CO2Incubator is cultivated 72 hours.Use trypsinization centrifuge cell, use CMC model
Base re-suspended cell, takes a small amount of cell suspension, adds trypan blue, is placed on cell viability calculating instrument and carries out carefully
Born of the same parents' viability examination.Remaining cell carries out passage or other experiments.Cell viability testing result is shown in Fig. 9.
As shown in Figure 9, test group cell brings back to life vigor and is higher than control group, shows the present embodiment recovery medium
Dental pulp mescenchymal stem cell is conducive to bring back to life raising and the cell proliferation of vigor, it is possible to preferably to keep dental pulp
The biological property of mescenchymal stem cell.
(3) the stem cell surface mark after flow cytomery recovery
After recovering, cell confluency degree reaches 80%-90%, with 0.25% trypsinization centrifuge cell, and counts
After often pipe add 2 × 105Cell number, dye solution washes 1 time, and 1000rpm is centrifuged 5min；Abandon supernatant, use
Dye solution piping and druming mixing cell；Add CD73, CD90, CD105, CD34, CD45 and
The each 2 μ L of HLA-DRA antibody, and set a pipe as blank；At 4 DEG C, lucifuge reaction 15-20min；
Dye solution is washed once, and 1000rpm is centrifuged 5min；Directly the cell of mark abandons supernatant, and lucifuge adds
The sample-loading buffer of 500 μ L, mixing, with 200 eye mesh screen filtration cell samples, flow cytomery is thin
Cellular surface antigen.The result of the test of test group is shown in Figure 10, and the result of the test of control group is shown in Figure 11.
From Figure 10,11, test group use the present invention provide recovery medium to frozen mesenchyma
After stem cell carries out recovery cultivation, negative surface marker CD34, CD45, HLA-DR are all to present feminine gender,
Mescenchymal stem cell surface marker CD73, CD90 simultaneously, CD105 all present the positive.After recovery is described
Dental pulp mescenchymal stem cell still keeps the biological property of mescenchymal stem cell.
(4) after recovery, cell induction Osteoblast Differentiation is identified
After to be tested group of recovery, cell confluency degree reaches 80%-90%, with 0.25% trypsinization centrifuge cell,
According to 5 × 103Cell/cm2It is inoculated in six orifice plates, after adding complete medium cultivation 24h, adds skeletonization and lure
Lead culture medium and carry out Fiber differentiation, changed liquid every 2-3 days, after 28 days, carry out Alizarin red staining, result and Fig. 4
Similar.Visible, after recovery mescenchymal stem cell, after osteogenic induction 4 weeks, can by Alizarin red staining
See calcium scoring, illustrate that the dental pulp mescenchymal stem cell after recovery medium of the present invention is recovered still is protected
Hold the potential to Osteoblast Differentiation.
(5) after recovery, mescenchymal stem cell induces into fat differentiation qualification
After to be tested group of recovery, cell confluency degree reaches 80%-90%, with 0.25% trypsinization centrifuge cell,
After collecting cell, and according to 5 × 103Cell/cm2It is inoculated in six orifice plates, after adding complete medium 24h,
Add adipogenic induction culture medium to cultivate.Adipogenic induction culture medium include basal medium DMEM, 10%
Hyclone, 0.5mM IBMX, 1 μM of dexamethasone, 100 μMs of Indomethacins, 5 μ g/mL pancreas islet
Element, 2mm/L glutamine etc..Within every three days, change liquid.Carry out oil red O stain after surrounding, identify that fat drips formation
Situation, result is similar to Fig. 5.Visible, after recovery mescenchymal stem cell, after adipogenic induction 4 weeks,
By oil red O stain red color visible oil droplet, the dental pulp mesenchyma after recovery medium of the present invention is recovered is described
Stem cell remains in that the potential to Adipose Differentiation.
The above is only the preferred embodiment of the present invention, it is noted that general for the art
For logical technical staff, under the premise without departing from the principles of the invention, it is also possible to make some improvement and profit
Decorations, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (10)
1. the recovery medium of a dental pulp mescenchymal stem cell, it is characterised in that described recovery medium
Preparation method be: take dental pulp mescenchymal stem cell, after using complete medium to carry out the first cultivation, then
After using serum free medium to carry out the second cultivation, collect supernatant, it is thus achieved that recovery medium.
Recovery medium the most according to claim 1, it is characterised in that described first cultivate time
Between be 24～72h.
Recovery medium the most according to claim 1, it is characterised in that the described first tooth cultivated
The inoculum density of bone marrow-drived mesenchymal stem is (5～10) × 103cell/cm2。
Recovery medium the most according to claim 1, it is characterised in that described serum free medium
For DMEM/F12 culture medium.
Recovery medium the most according to claim 1, it is characterised in that described second cultivate time
Between be 24～72h.
Recovery medium the most according to claim 1, it is characterised in that after described collection supernatant
Also include the step being centrifuged.
Recovery medium the most according to claim 6, it is characterised in that described being centrifuged is
1000～2000rpm are centrifuged 5～10min.
8. according to the recovery medium described in claim 6 or 7, it is characterised in that described centrifugal after also
Step including filtration sterilization.
9. the recovery cultural method of a dental pulp mescenchymal stem cell, it is characterised in that described employing right
Require that frozen dental pulp mescenchymal stem cell is cultivated in the recovery medium recovery according to any one of 1 to 8.
Recovery cultural method the most according to claim 9, it is characterised in that described recovery is cultivated
Method is: take frozen dental pulp mescenchymal stem cell, after incubation, uses in claim 1 to 8 and appoints
The dental pulp mescenchymal stem cell after described incubation is cultivated in one described recovery medium recovery.
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| CN111727961A (en) * | 2020-08-10 | 2020-10-02 | 医微细胞生物技术(广州)有限公司 | Dental pulp stem cell cryopreservation liquid and cryopreservation method thereof |
| WO2020241132A1 (en) * | 2019-05-29 | 2020-12-03 | パナジー株式会社 | Cell proliferation method, cell proliferation agent, and medium for cell proliferation |
| CN114015648A (en) * | 2021-10-20 | 2022-02-08 | 成都拜美森生物科技有限公司 | High-performance adipose-derived mesenchymal stem cell solution and preparation method and application thereof |
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| CN105238750A (en) * | 2015-11-17 | 2016-01-13 | 广州赛莱拉干细胞科技股份有限公司 | Method for resuscitating umbilical cord mesenchymal stem cells |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107083358A (en) * | 2017-06-12 | 2017-08-22 | 广州赛莱拉干细胞科技股份有限公司 | A kind of cell culture medium and the application in umbilical cord mesenchymal stem cells culture |
| WO2020241132A1 (en) * | 2019-05-29 | 2020-12-03 | パナジー株式会社 | Cell proliferation method, cell proliferation agent, and medium for cell proliferation |
| JP2020195363A (en) * | 2019-05-29 | 2020-12-10 | パナジー株式会社 | Cell proliferation method, cell proliferation agent, and medium for cell proliferation |
| CN111727961A (en) * | 2020-08-10 | 2020-10-02 | 医微细胞生物技术(广州)有限公司 | Dental pulp stem cell cryopreservation liquid and cryopreservation method thereof |
| CN111727961B (en) * | 2020-08-10 | 2020-12-01 | 医微细胞生物技术(广州)有限公司 | Dental pulp stem cell cryopreservation liquid and cryopreservation method thereof |
| CN114015648A (en) * | 2021-10-20 | 2022-02-08 | 成都拜美森生物科技有限公司 | High-performance adipose-derived mesenchymal stem cell solution and preparation method and application thereof |
| CN114015648B (en) * | 2021-10-20 | 2024-02-20 | 成都拜美森生物科技有限公司 | High-performance adipose-derived mesenchymal stem cell solution and preparation method and application thereof |
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Application publication date: 20160817 |