CN106417250A - Dental pulp mesenchymal stem cell cryopreservation solution and cryopreservation method thereof - Google Patents
Dental pulp mesenchymal stem cell cryopreservation solution and cryopreservation method thereof Download PDFInfo
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- CN106417250A CN106417250A CN201610609538.1A CN201610609538A CN106417250A CN 106417250 A CN106417250 A CN 106417250A CN 201610609538 A CN201610609538 A CN 201610609538A CN 106417250 A CN106417250 A CN 106417250A
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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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- C—CHEMISTRY; METALLURGY
- 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
- C12N5/0664—Dental pulp stem cells, Dental follicle stem cells
Abstract
The invention relates to the technical field of cell culture, and particularly relates to a dental pulp mesenchymal stem cell cryopreservation solution and a cryopreservation method thereof. The cryopreservation solution is a conditioned medium containing DMSO, FBS and dental pulp mesenchymal stem cells; a preparation method of the dental pulp mesenchymal stem cell conditioned medium includes the steps of conducting cell culture of dental pulp mesenchymal stem cells by using a cell culture medium, and after cell confluence degree reaches 80%-90%, collecting supernatant in the cell culture medium to obtain the dental pulp mesenchymal stem cell conditioned medium. The cryopreservation solution is adopted for cryopreservation of the dental pulp mesenchymal stem cells, the damage effect on the dental pulp mesenchymal stem cells by the cryopreservation solution is thus greatly reduced, cell viability of the dental pulp mesenchymal stem cells in cryopreservation process and thawing and waking process are improved, and biological characteristics of the dental pulp mesenchymal stem cells can be better maintained.
Description
Technical field
The present invention relates to technical field of cell culture, particularly to a kind of frozen stock solution of dental pulp mescenchymal stem cell and its jelly
Deposit method.
Background technology
Periodontitis are the chronic infective involving four kinds of Periodontal Supporting Tissue (gingiva, periodontal membrane, alveolar bone and cementum)
Disease, often causes the inflammatory destruction of Periodontal Supporting Tissue.More common after 35 years old.Commonly encountered diseases are because bacterial plaque, tartar, wound
The occlusion of wound property, food impaction, ill fitting prosthesis, mouth breathing etc..Periodontitis have four big features, i.e. periodontal bag formation, bag wall
Inflammation, frontal resorption, tooth gradually loosen.Serious periodontitis can cause loss of tooth, thus leading to masticatory function low
And cause dyspepsia and gastrointestinal disease, affect physical and mental health.
Essentially consisting in of periodontitis for the treatment of diminishes inflammation, and promotes the regeneration of destroyed periodontal tissue, is just recovering tooth
Chang Gongneng.Clinical frequently with periodontitis treatment method include：Periodental non-surgical treatment (scaling, scrape control, root planing), periodontal turns over
Lobe art 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 a kind of method of prevention and treatment odontopathy.Use
The pigment of attachment on supragingival calculuss, bacterial plaque and facing removed by scaling apparatus, and polishes facing, is to prevent bacterial plaque and tartar from sinking again
Long-pending, prevent and treat the measure of periodontal disease.Also should be by a part of subgingival calculuss being connected with supragingival calculuss in gingival sulcus also in scaling
And dispose.Different according to apparatus used, supragingival scaling is divided into hand instrument scaling method and ultrasonic cavitron scaling method.
For gingivitis patients, make a scaling within every 6～12 months, can effectively safeguard periodontal health.Subgingival curettage, that is, root face is flat
Whole art, it is that subgingival debridement apparatus by hand stretches into remove in periodontal pocket and is attached on root face in periodontal pocket and embedded cementum
Interior subgingival calculuss and bacterial plaque, and strike off the pathological changes cementum that root surface is subject to endotoxin contamination, thus being formed smooth, cleaning
Root face, makes root face have biocompatible surface, is conducive to attachment and the new life of periodontal tissue.Root planing bel not applied to be good for
Health periodontal loci, in order to avoid lead to periodontal attachment loss.
Periodontal flap surgery：Refer to, using different operative incisions, the separate tissue of gingiva and lower section form gingiva tissue
Lobe, exposes root face and the alveolar bone of diseased region, provides debridement approach and visuality.After striking off pathological tissues and bacterial plaque tartar, will
Gingival flap resets and goes up in place and suture, and reaches the purpose eliminating periodontal pocket or making periodontal pocket shoal.
Paradenlal tissue regeneration art：In the alveolar bone area implantable artificial bone having lacked, and cover special biological guiding film,
Grow on root face, thus reaching periodontal membrane, alveolar bone and cemental regeneration with making the cell selective on periodontal membrane.Pass through
This bone is planted and biomembranous technology, recovers alveolar bone, gingiva and the other periodontal tissue having destroyed, completes the weight of tooth
Newly stable.
Above method cannot repair the periodontal attachment of damage and alveolus is organized it is impossible to meet at present clinically to oral and maxillofacial surgery
Portion's Repair of tissue defect regeneration and the requirement of function, profile recovery, next periodontitis treatment usually controls bacterial plaque to give birth to by antibiosis
Long, carry out the Drug therapy of whole body and local.But antibiotic side effect is more, pathogenic microorganism also can produce drug resistance.
Dental pulp stem cell (dental pulp stem cells, DPSCs) is the class being present in tooth pulp tissue
There is self renewal, multiplication capacity by force, and the mescenchymal stem cell of Multidirectional Differentiation ability.Dental pulp stem cell has Multidirectional Differentiation
Potential, it except the extracellular of Mineral nodules ability can be formed, through different cytokines induction additionally it is possible to be divided into fat
The cell set type such as fat, bone, cartilage, muscle, blood vessel endothelium, liver, nerve.It is reported that, dental pulp stem cell is in periodontitis treatment
Good repair can be played, mechanism of action is：Defect cell, secrete cytokines locally and are divided into by implantation, become
Change stem cell to local, suppress local inflammation, promote local vascular newborn.
Be related to frozen, the resuscitation technique of dental pulp stem cell in the clinical practice of dental pulp stem cell, cell cryopreservation be by
The cell of 37 DEG C of grown cultures, adds nutritional labeling and anti-frost protection agent DMSO, by cell long-period by way of gradually lowering the temperature
Process in liquid nitrogen for the Cryopreservation.When cell thaws rapidly to room temperature process, activity and the guarantor of cell still can be recovered
Hold good biological property.At present, the frozen usual employing dimethyl sulfoxide (DMSO) of cell stem cell is carried out frozen, also
The culture medium of general commercial or serum can be adopted to carry out cell cryopreservation.But these frozen stock solutions cell frozen during still
Dental pulp stem cell can cannot be caused with avoiding with certain damaging it is impossible to ensure the vigor of stem cell, cell proliferation rate is relatively low, shadow
The clinic ringing stem cell utilizes.Accordingly, it would be desirable to exploitation is a kind of can preferably maintain the frozen of dental pulp mescenchymal stem cell vigor
Liquid and its cryopreservation methods.
Content of the invention
In view of this, the invention provides a kind of frozen stock solution of dental pulp mescenchymal stem cell and its cryopreservation methods.This is frozen
Liquid energy enough preferably maintains dental pulp mescenchymal stem cell vigor.
In order to realize foregoing invention purpose, the present invention provides technical scheme below：
The invention provides a kind of frozen stock solution of dental pulp mescenchymal stem cell, do thin including DMSO, FBS and dental pulp mesenchyme
The conditioned medium of born of the same parents；
The preparation method of the conditioned medium of dental pulp mescenchymal stem cell is：Dental pulp mescenchymal stem cell adopts cell culture
Base carries out cell culture, treats that cell confluency degree reaches 80%～90%, collects cell culture medium supernatant, obtains dental pulp mesenchyme
The conditioned medium of stem cell.
Preferably, the volume ratio of the conditioned medium of DMSO, FBS and dental pulp mescenchymal stem cell is (1～3)：(3～
5)：(4～6).
Preferably, the volume ratio of the conditioned medium of DMSO, FBS and dental pulp mescenchymal stem cell is 1：4：5.
In some embodiments that the present invention provides, cell culture medium is the DMEM/F12 culture medium containing 10%FBS.
Preferably, the dental pulp mescenchymal stem cell inoculum density of cell culture is (5～10) × 103/cm2.
Preferably, the dental pulp mescenchymal stem cell inoculum density of cell culture is 7 × 103/cm2.
In some embodiments that the present invention provides, cell culture is in 37 DEG C, 5%CO2Under the conditions of cultivate.
Preferably, also including the step being centrifuged after collecting cell culture medium supernatant.
Preferably, be centrifuged being centrifuged 5～10min for 1000～2000rpm.
In some embodiments that the present invention provides, it is centrifuged and is centrifuged 5min for 1000rpm.
Preferably, the step also including filtration sterilization after centrifugation.
In some embodiments that the present invention provides, the aperture for the filter membrane of filtration sterilization is 0.22 μm.
Present invention also offers a kind of cryopreservation methods of dental pulp mescenchymal stem cell, frozen using the frozen stock solution that the present invention provides
Deposit dental pulp mescenchymal stem cell.
Preferably, in terms of mL/cell, the ratio of frozen stock solution and dental pulp mescenchymal stem cell is 1：(1～2) × 106.
In some embodiments that the present invention provides, in terms of mL/cell, the ratio of frozen stock solution and dental pulp mescenchymal stem cell
For 1：1×106.
Preferably, frozen temperature≤- 80 DEG C.
The invention provides a kind of frozen stock solution of dental pulp mescenchymal stem cell and its cryopreservation methods.This frozen stock solution includes
DMSO, FBS and the conditioned medium of dental pulp mescenchymal stem cell；The preparation method of the conditioned medium of dental pulp mescenchymal stem cell
For：Dental pulp mescenchymal stem cell carries out cell culture using cell culture medium, treats that cell confluency degree reaches 80%～90%, collects
Cell culture medium supernatant, obtains the conditioned medium of dental pulp mescenchymal stem cell.Beneficial effects of the present invention are：Using this
Bright frozen stock solution carries out frozen to dental pulp mescenchymal stem cell, greatly reduces frozen stock solution and the damage of dental pulp mescenchymal stem cell is made
With being conducive to the raising of dental pulp mescenchymal stem cell cell viability in frozen and resuscitation process, can preferably keep dental pulp
The biological property of mescenchymal stem cell.
Brief description
Fig. 1 shows the flow cytometer detection result of the dental pulp mescenchymal stem cell after recovery in embodiment 1；Wherein Fig. 1-1 shows CD73
Testing result, Fig. 1-2 shows CD90 testing result, and Fig. 1-3 shows CD105 testing result, and Fig. 1-4 shows CD34 testing result, and Fig. 1-5 shows
CD45 testing result, Fig. 1-6 shows HLA-DR testing result；
Fig. 2 shows the induced osteogenesis differentiation qualification result of the dental pulp mescenchymal stem cell after recovery in embodiment 1；
Fig. 3 shows that the induction of the dental pulp mescenchymal stem cell after recovery in embodiment 1 becomes fat differentiation qualification result.
Fig. 4 shows the flow cytometer detection result of the dental pulp mescenchymal stem cell after recovery in embodiment 2；Wherein Fig. 1-1 shows CD73
Testing result, Fig. 1-2 shows CD90 testing result, and Fig. 1-3 shows CD105 testing result, and Fig. 1-4 shows CD34 testing result, and Fig. 1-5 shows
CD45 testing result, Fig. 1-6 shows HLA-DR testing result；
Fig. 5 shows the flow cytometer detection result of the dental pulp mescenchymal stem cell after recovery in embodiment 3；Wherein Fig. 1-1 shows CD73
Testing result, Fig. 1-2 shows CD90 testing result, and Fig. 1-3 shows CD105 testing result, and Fig. 1-4 shows CD34 testing result, and Fig. 1-5 shows
CD45 testing result, Fig. 1-6 shows HLA-DR testing result；
Fig. 6 shows the cell viability detection figure that embodiment 1 mesenchymal stem cells MSCs are recovered after frozen through different frozen stock solutions, its
Middle experimental group is that the frozen stock solution of the embodiment of the present invention 1 mesenchymal stem cells MSCs is frozen, and matched group is that standard frozen stock solution is frozen.
Specific embodiment
The invention discloses a kind of frozen stock solution of dental pulp mescenchymal stem cell and its cryopreservation methods, those skilled in the art can
To use for reference present disclosure, it is suitably modified technological parameter and realizes.Specifically, all similar replacements and change are to this
It is it will be apparent that they are considered as including in the present invention for skilled person.The method of the present invention and application are
It is described by preferred embodiment, related personnel substantially can be to herein in without departing from present invention, spirit and scope
Described methods and applications are modified or suitably change and combine, and to realize and to apply the technology of the present invention.
In the frozen stock solution of dental pulp mescenchymal stem cell that the present invention provides and its cryopreservation methods, biomaterial used all can be by
Market is buied.
With reference to embodiment, the present invention is expanded on further：
Embodiment 1
(1) collection of the culture of dental pulp mescenchymal stem cell and conditioned medium
(1) culture of dental pulp mescenchymal stem cell
Collect the healthy tooth that comes off with servant and extract for 30 years old, PBS dental surface dirt, with clamp schizodont tooth,
Exposed Pulp is organized；Grip pulp tissue with aseptic nipper.Shred, place the 3-5g/ adding 10 times of volumes in 50mL centrifuge tube
L type i collagen enzyme, is sufficiently mixed after sealing uniformly, is transferred in Tempeerature-constant air shaking table, 37 DEG C, a 200R digestion 10-20min left side
The right side, adds isopyknic complete medium to terminate digestion, repeatedly blows and beats discrete cellular agglomerate, by 70 μm of cell screen clothes mistake
Filter, to obtain single discrete cell, 1000rpm is centrifuged 5min.Abandon supernatant, precipitated 1 time with 30mL PBS,
1000rpm is centrifuged 5min.Precipitation DMEM/F12+10%FBS cultivates resuspended, with 0.5～1 × 104/cm2It is seeded to six orifice plates
In, every hole adds 2mL growth medium, and (both are whole to add epidermal growth factor EGF and basic fibroblast growth factor bFGF
Concentration be 10ng/mL), mix cell suspension, be put in 37 DEG C, humidity be 95% CO2 gas incubator in cultivate.Every 3d changes
Liquid 1 time, 7-14d about under the microscope it is observed that clone formation, when cell growth is converged to 80%-90%,
0.125% trypsin digestion and cell, carries out Secondary Culture.With the resuspended precipitation of complete medium after centrifugation, 7 × 103/cm2Cell
Continue culture in density inoculated and cultured ware.P3-P5 is taken to 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 inoculated and cultured ware,
DMEM/F12+10%FBS culture medium is cultivated, and treats that cell confluency degree reaches 80%-90%, collects cell culture medium supernatant,
Supernatant is sub-packed in 50mL centrifuge tube, 1000～2000rpm is centrifuged 5～10min.Remove precipitation, collect supernatant.With disposable
Syringe draws conditioned medium, degerming with 0.22 μm of membrane filtration, is collected in sterile culture flask.
(2) dental pulp mescenchymal stem cell is frozen
When dental pulp mescenchymal stem cell degree of converging reaches 80%-90%, with PBS 2 times, add 0.25% pancreatin digestion
Cell 2min, after cell rounding, adds the culture medium containing serum to terminate digestion, is centrifuged 5-10min with 1000-1500rpm, goes
Clearly, add FBS re-suspended cell precipitation, go 20-50 μ l cell suspension to carry out cell counting, every 1 × 106Cell 1mL frozen stock solution
Carry out frozen (formula of 1mL cells frozen storing liquid be 0.1mL DMSO, 0.4mL FBS, 0.5mL dental pulp mescenchymal stem cell condition
Culture medium).Cell is placed in cryopreservation tube, is put in the program temperature reduction box containing isopropanol, put into -80 DEG C overnight after, you can proceed to
Carry out Long-term Cryopreservation in liquid nitrogen.
(3) recovery of dental pulp mescenchymal stem cell
Frozen dental pulp mescenchymal stem cell is taken out from liquid nitrogen, is placed in 35-42 DEG C of water-bath, concussion incubates
60 seconds, after cell thawing, immediately frozen stock solution is added in the conditioned medium of 5 times of volumes, takes a small amount of cell suspension, add platform
Expect indigo plant, be placed on cell viability calculating instrument and carry out cell viability detection, remaining cell suspension, with 1000 centrifugation 5 minutes, removes supernatant,
With step () (2nd) partly middle acquisition conditioned medium (conditioned medium mentioned below is this conditioned medium) weight
Outstanding precipitation；Gently piping and druming mixes, and according to 7 × 103/cm2In cell density inoculated and cultured ware, cultivated with conditioned medium.Place
At 37 DEG C, 5%CO2After incubator stands 24 hours, change conditioned medium and continue to cultivate, daily observation of cell amplification situation,
Cell carries out continuing culture 48 hours, digests centrifuge cell with pancreatin, uses conditioned medium re-suspended cell, takes a small amount of cell to hang
Liquid, add trypan blue, be placed on cell viability calculating instrument and carry out cell viability detection, remaining cell carry out passage or other
Experiment.
(4) stem cell surface mark after flow cytomery recovery
After recovering, cell confluency degree reaches 80%-90%, digests centrifuge cell with 0.25% pancreatin, and often manages after counting
Add 2 × 105Cell number, dye solution is washed 1 time, and 1000rpm is centrifuged 5min；Abandon supernatant, blown and beaten with dye solution and mix
Cell；Add each 2 μ L of CD73, CD90, CD105, CD34, CD45 and HLA-DR antibody, and set a pipe as blank；At 4 DEG C
Under, lucifuge reacts 15-20min；Dye solution is washed once, and 1000rpm is centrifuged 5min；Directly the cell of labelling abandons supernatant, keeps away
Light adds the sample-loading buffer of 500 μ L, mixes, and with 200 eye mesh screen filtration cell samples, flow cytomery cell surface resists
Former.Testing result is shown in Fig. 1.
As seen from Figure 1, mescenchymal stem cell feminine gender surface CD34, CD45, HLA-DR all assumes feminine gender, and mesenchyme is done simultaneously
Cell surface marker thing CD73, CD90, CD105 all assume the positive.Between illustrating that recovery backteeth bone marrow-drived mesenchymal stem still keeps
The biological property of mesenchymal stem cells.
(5) cell induction Osteoblast Differentiation identification after cryopreservation resuscitation
After recovering, cell confluency degree reaches 80%-90%, digests centrifuge cell with 0.25% pancreatin, according to 1 × 103Carefully
Born of the same parents/cm2It is inoculated in six orifice plates, after adding complete medium culture 24h, add Osteogenic Induction Medium to carry out inducing culture,
Changed liquid every 2 days, after 28 days, carry out Alizarin red staining, result is shown in Fig. 2.
As shown in Figure 2, the mescenchymal stem cell after recovery is after osteogenic induction 3 weeks, by the visible calcification of Alizarin red staining
Tuberosity, illustrates that the dental pulp mescenchymal stem cell after conditioned medium cryopreservation resuscitation remains in that the potential to Osteoblast Differentiation.
(6) after cryopreservation resuscitation, mescenchymal stem cell induces into fat differentiation identification
After recovering, cell confluency degree reaches 80%-90%, digests centrifuge cell with 0.25% pancreatin, after collecting cell,
And according to 1 × 103Cell/cm2It is inoculated in six orifice plates, adds complete medium, after 24h, add adipogenic induction culture medium to carry out
Culture.Adipogenic induction culture medium include basal medium DMEM, 10% hyclone, 0.5mM IBMX, 1 μM of dexamethasone, 100
μM indomethacin, 5 μ g/mL insulins, 2mm/L L-Glutamine etc..Change liquid within every three days.Carry out oil red O stain after three weeks, identify fat
Drip formational situation, result is shown in Fig. 3.
From the figure 3, it may be seen that the mescenchymal stem cell after recovery is after adipogenic induction 3 weeks, by oil red O stain red color visible oil
Drip, illustrate that the dental pulp mescenchymal stem cell after conditioned medium cryopreservation resuscitation remains in that the potential to Adipose Differentiation.
Embodiment 2
(1) collection of the culture of dental pulp mescenchymal stem cell and conditioned medium
(1) culture of dental pulp mescenchymal stem cell
Collect the healthy tooth that comes off with servant and extract for 30 years old, PBS dental surface dirt, with clamp schizodont tooth,
Exposed Pulp is organized；Grip pulp tissue with aseptic nipper.Shred, place the 3-5g/ adding 10 times of volumes in 50mL centrifuge tube
L type i collagen enzyme, is sufficiently mixed after sealing uniformly, is transferred in Tempeerature-constant air shaking table, 37 DEG C, a 200R digestion 10-20min left side
The right side, adds isopyknic complete medium to terminate digestion, repeatedly blows and beats discrete cellular agglomerate, by 70 μm of cell screen clothes mistake
Filter, to obtain single discrete cell, 1000rpm is centrifuged 5min.Abandon supernatant, precipitated 1 time with 30mL PBS,
1000rpm is centrifuged 5min.Precipitation DMEM/F12+10%FBS cultivates resuspended, with 0.5～1 × 104/cm2It is seeded to six orifice plates
In, every hole adds 2mL growth medium, and (both are whole to add epidermal growth factor EGF and basic fibroblast growth factor bFGF
Concentration be 10ng/mL), mix cell suspension, be put in 37 DEG C, humidity be 95% CO2 gas incubator in cultivate.Every 3d changes
Liquid 1 time, 7-14d about under the microscope it is observed that clone formation, when cell growth is converged to 80%-90%,
0.125% trypsin digestion and cell, carries out Secondary Culture.With the resuspended precipitation of complete medium after centrifugation, 7 × 103/cm2Cell
Continue culture in density inoculated and cultured ware.P3-P5 is taken to 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 inoculated and cultured ware,
DMEM/F12+10%FBS culture medium is cultivated, and treats that cell confluency degree reaches 80%-90%, collects cell culture medium supernatant,
Supernatant is sub-packed in 50mL centrifuge tube, 1000～2000rpm is centrifuged 5～10min.Remove precipitation, collect supernatant.With disposable
Syringe draws conditioned medium, degerming with 0.22 μm of membrane filtration, is collected in sterile culture flask.
(2) dental pulp mescenchymal stem cell is frozen
When dental pulp mescenchymal stem cell degree of converging reaches 80%-90%, with PBS 2 times, add 0.25% pancreatin digestion
Cell 2min, after cell rounding, adds the culture medium containing serum to terminate digestion, is centrifuged 5-10min with 1000-1500rpm, goes
Clearly, add FBS re-suspended cell precipitation, go 20-50 μ l cell suspension to carry out cell counting, every 1 × 106Cell 1mL frozen stock solution
Carry out frozen (formula of cells frozen storing liquid be 0.2mL DMSO, 0.5mL FBS, 0.4mL dental pulp mescenchymal stem cell CMC model
Base).Cell is placed in cryopreservation tube, is put in the program temperature reduction box containing isopropanol, put into -80 DEG C overnight after, you can proceed to liquid nitrogen
In carry out Long-term Cryopreservation.
(3) recovery of dental pulp mescenchymal stem cell
Frozen dental pulp mescenchymal stem cell is taken out from liquid nitrogen, is placed in 35-42 DEG C of water-bath, concussion incubates
60 seconds, after cell thawing, immediately frozen stock solution is added in the conditioned medium of 5 times of volumes, takes a small amount of cell suspension, add platform
Expect indigo plant, be placed on cell viability calculating instrument and carry out cell viability detection, remaining cell suspension, with 1000 centrifugation 5 minutes, removes supernatant,
With step () (2nd) partly middle acquisition conditioned medium (conditioned medium mentioned below is this conditioned medium) weight
Outstanding precipitation；Gently piping and druming mixes, and according to 7 × 103/cm2In cell density inoculated and cultured ware, cultivated with conditioned medium.Place
At 37 DEG C, 5%CO2After incubator stands 24 hours, change conditioned medium and continue to cultivate, daily observation of cell amplification situation,
Cell carries out continuing culture 48 hours, digests centrifuge cell with pancreatin, uses conditioned medium re-suspended cell, takes a small amount of cell to hang
Liquid, add trypan blue, be placed on cell viability calculating instrument and carry out cell viability detection, remaining cell carry out passage or other
Experiment.
(4) stem cell surface mark after flow cytomery recovery
After recovering, cell confluency degree reaches 80%-90%, digests centrifuge cell with 0.25% pancreatin, and often manages after counting
Add 2 × 105Cell number, dye solution is washed 1 time, and 1000rpm is centrifuged 5min；Abandon supernatant, blown and beaten with dye solution and mix
Cell；Add each 2 μ L of CD73, CD90, CD105, CD34, CD45 and HLA-DR antibody, and set a pipe as blank；At 4 DEG C
Under, lucifuge reacts 15-20min；Dye solution is washed once, and 1000rpm is centrifuged 5min；Directly the cell of labelling abandons supernatant, keeps away
Light adds the sample-loading buffer of 500 μ L, mixes, and with 200 eye mesh screen filtration cell samples, flow cytomery cell surface resists
Former.Testing result is shown in Fig. 4.
From fig. 4, it can be seen that mescenchymal stem cell feminine gender surface CD34, CD45, HLA-DR all assume feminine gender, mesenchyme is done simultaneously
Cell surface marker thing CD73, CD90, CD105 all assume the positive.Between illustrating that recovery backteeth bone marrow-drived mesenchymal stem still keeps
The biological property of mesenchymal stem cells.
(5) cell induction Osteoblast Differentiation identification after cryopreservation resuscitation
After recovering, cell confluency degree reaches 80%-90%, digests centrifuge cell with 0.25% pancreatin, according to 1 × 103Carefully
Born of the same parents/cm2It is inoculated in six orifice plates, after adding complete medium culture 24h, add Osteogenic Induction Medium to carry out inducing culture,
Changed liquid every 2 days, after 28 days, carry out Alizarin red staining, result is similar to Fig. 2.
It can be seen that, the mescenchymal stem cell after recovery after osteogenic induction 3 weeks, by the visible calcium scoring of Alizarin red staining,
Illustrate that the dental pulp mescenchymal stem cell after conditioned medium cryopreservation resuscitation remains in that the potential to Osteoblast Differentiation.
(6) after cryopreservation resuscitation, mescenchymal stem cell induces into fat differentiation identification
After recovering, cell confluency degree reaches 80%-90%, digests centrifuge cell with 0.25% pancreatin, after collecting cell,
And according to 1 × 103Cell/cm2It is inoculated in six orifice plates, adds complete medium, after 24h, add adipogenic induction culture medium to carry out
Culture.Adipogenic induction culture medium include basal medium DMEM, 10% hyclone, 0.5mM IBMX, 1 μM of dexamethasone, 100
μM indomethacin, 5 μ g/mL insulins, 2mm/L L-Glutamine etc..Change liquid within every three days.Carry out oil red O stain after 3 weeks, identify fat
Drip formational situation, result is similar to Fig. 3.
It can be seen that, the mescenchymal stem cell after recovery, after adipogenic induction 3 weeks, by oil red O stain red color visible oil droplet, is said
Dental pulp mescenchymal stem cell after the bright cryopreservation resuscitation through conditioned medium remains in that the potential to Adipose Differentiation.
Embodiment 3
(1) collection of the culture of dental pulp mescenchymal stem cell and conditioned medium
(1) culture of dental pulp mescenchymal stem cell
Collect the healthy tooth that comes off with servant and extract for 30 years old, PBS dental surface dirt, with clamp schizodont tooth,
Exposed Pulp is organized；Grip pulp tissue with aseptic nipper.Shred, place the 3-5g/ adding 10 times of volumes in 50mL centrifuge tube
L type i collagen enzyme, is sufficiently mixed after sealing uniformly, is transferred in Tempeerature-constant air shaking table, 37 DEG C, a 200R digestion 10-20min left side
The right side, adds isopyknic complete medium to terminate digestion, repeatedly blows and beats discrete cellular agglomerate, by 70 μm of cell screen clothes mistake
Filter, to obtain single discrete cell, 1000rpm is centrifuged 5min.Abandon supernatant, precipitated 1 time with 30mL PBS,
1000rpm is centrifuged 5min.Precipitation DMEM/F12+10%FBS cultivates resuspended, with 0.5～1 × 104/cm2It is seeded to six orifice plates
In, every hole adds 2mL growth medium, and (both are whole to add epidermal growth factor EGF and basic fibroblast growth factor bFGF
Concentration be 10ng/mL), mix cell suspension, be put in 37 DEG C, humidity be 95% CO2 gas incubator in cultivate.Every 3d changes
Liquid 1 time, 7-14d about under the microscope it is observed that clone formation, when cell growth is converged to 80%-90%,
0.125% trypsin digestion and cell, carries out Secondary Culture.With the resuspended precipitation of complete medium after centrifugation, 7 × 103/cm2Cell
Continue culture in density inoculated and cultured ware.P3-P5 is taken to 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 inoculated and cultured ware,
DMEM/F12+10%FBS culture medium is cultivated, and treats that cell confluency degree reaches 80%-90%, collects cell culture medium supernatant,
Supernatant is sub-packed in 50mL centrifuge tube, 1000～2000rpm is centrifuged 5～10min.Remove precipitation, collect supernatant.With disposable
Syringe draws conditioned medium, degerming with 0.22 μm of membrane filtration, is collected in sterile culture flask.
(2) dental pulp mescenchymal stem cell is frozen
When dental pulp mescenchymal stem cell degree of converging reaches 80%-90%, with PBS 2 times, add 0.25% pancreatin digestion
Cell 2min, after cell rounding, adds the culture medium containing serum to terminate digestion, is centrifuged 5-10min with 1000-1500rpm, goes
Clearly, add FBS re-suspended cell precipitation, go 20-50 μ l cell suspension to carry out cell counting, every 1 × 106Cell 1mL frozen stock solution
Carry out frozen (formula of cells frozen storing liquid be 0.3mL DMSO, 0.3mL FBS, 0.3mL dental pulp mescenchymal stem cell CMC model
Base).Cell is placed in cryopreservation tube, is put in the program temperature reduction box containing isopropanol, put into -80 DEG C overnight after, you can proceed to liquid nitrogen
In carry out Long-term Cryopreservation.
(3) recovery of dental pulp mescenchymal stem cell
Frozen dental pulp mescenchymal stem cell is taken out from liquid nitrogen, is placed in 35-42 DEG C of water-bath, concussion incubates
60 seconds, after cell thawing, immediately frozen stock solution is added in the conditioned medium of 5 times of volumes, takes a small amount of cell suspension, add platform
Expect indigo plant, be placed on cell viability calculating instrument and carry out cell viability detection, remaining cell suspension, with 1000 centrifugation 5 minutes, removes supernatant,
With step () (2nd) partly middle acquisition conditioned medium (conditioned medium mentioned below is this conditioned medium) weight
Outstanding precipitation；Gently piping and druming mixes, and according to 7 × 103/cm2In cell density inoculated and cultured ware, cultivated with conditioned medium.Place
At 37 DEG C, 5%CO2After incubator stands 24 hours, change conditioned medium and continue to cultivate, daily observation of cell amplification situation,
Cell carries out continuing culture 48 hours, digests centrifuge cell with pancreatin, uses conditioned medium re-suspended cell, takes a small amount of cell to hang
Liquid, add trypan blue, be placed on cell viability calculating instrument and carry out cell viability detection, remaining cell carry out passage or other
Experiment.
(4) stem cell surface mark after flow cytomery recovery
After recovering, cell confluency degree reaches 80%-90%, digests centrifuge cell with 0.25% pancreatin, and often manages after counting
Add 2 × 105Cell number, dye solution is washed 1 time, and 1000rpm is centrifuged 5min；Abandon supernatant, blown and beaten with dye solution and mix
Cell；Add each 2 μ L of CD73, CD90, CD105, CD34, CD45 and HLA-DR antibody, and set a pipe as blank；At 4 DEG C
Under, lucifuge reacts 15-20min；Dye solution is washed once, and 1000rpm is centrifuged 5min；Directly the cell of labelling abandons supernatant, keeps away
Light adds the sample-loading buffer of 500 μ L, mixes, and with 200 eye mesh screen filtration cell samples, flow cytomery cell surface resists
Former.Testing result is shown in Fig. 5.
As seen from Figure 5, mescenchymal stem cell feminine gender surface CD34, CD45, HLA-DR all assumes feminine gender, and mesenchyme is done simultaneously
Cell surface marker thing CD73, CD90, CD105 all assume the positive.Between illustrating that recovery backteeth bone marrow-drived mesenchymal stem still keeps
The biological property of mesenchymal stem cells.
(5) cell induction Osteoblast Differentiation identification after cryopreservation resuscitation
After recovering, cell confluency degree reaches 80%-90%, digests centrifuge cell with 0.25% pancreatin, according to 1 × 103Carefully
Born of the same parents/cm2It is inoculated in six orifice plates, after adding complete medium culture 24h, add Osteogenic Induction Medium to carry out inducing culture,
Changed liquid every 2 days, after 28 days, carry out Alizarin red staining, result is similar to Fig. 2.
It can be seen that, the mescenchymal stem cell after recovery after osteogenic induction 3 weeks, by the visible calcium scoring of Alizarin red staining,
Illustrate that the dental pulp mescenchymal stem cell after conditioned medium cryopreservation resuscitation remains in that the potential to Osteoblast Differentiation.
(6) after cryopreservation resuscitation, mescenchymal stem cell induces into fat differentiation identification
After recovering, cell confluency degree reaches 80%-90%, digests centrifuge cell with 0.25% pancreatin, after collecting cell,
And according to 1 × 103Cell/cm2It is inoculated in six orifice plates, adds complete medium, after 24h, add adipogenic induction culture medium to carry out
Culture.Adipogenic induction culture medium include basal medium DMEM, 10% hyclone, 0.5mM IBMX, 1 μM of dexamethasone, 100
μM indomethacin, 5 μ g/mL insulins, 2mm/L L-Glutamine etc..Change liquid within every three days.Carry out oil red O stain after 3 weeks, identify fat
Drip formational situation, result is similar to Fig. 3.
It can be seen that, the mescenchymal stem cell after recovery, after adipogenic induction 3 weeks, by oil red O stain red color visible oil droplet, is said
Dental pulp mescenchymal stem cell after the bright cryopreservation resuscitation through conditioned medium remains in that the potential to Adipose Differentiation.
Comparative example 1
Matched group (DMSO culture is basis set)：Degree of converging is taken to reach the dental pulp mescenchymal stem cell of 80-90%, with PBS 2
Secondary, add 0.25% trypsin digestion cell 2min, after cell rounding, add the culture medium containing serum to terminate digestion, with 1000-
1500rpm is centrifuged 5-10min, removes supernatant, adds FBS re-suspended cell precipitation, takes 20-50 μ L cell suspension to carry out cell counting,
Often (1-2) × 106Cell 1mL standard frozen stock solution carries out frozen.Cell is placed in cryopreservation tube and adds standard frozen stock solution, is put in containing different
In the program temperature reduction box of propanol, put into -80 DEG C overnight after, you can proceed to and in liquid nitrogen, carry out Long-term Cryopreservation.Recovery is taken out after half a year,
Calculate cell viability.Its Plays frozen stock solution is：Every 1mL standard frozen stock solution DMSO containing 0.1mL, 0.9mL FBS.
Experimental group (embodiment 1 frozen stock solution group)：Test method is with embodiment 1 operating procedure.
The cell viability result of matched group and experimental group is shown in Fig. 6.From Fig. 6 result, dental pulp mescenchymal stem cell passes through
Cell recovery vigor after the frozen stock solution (experimental group) of the embodiment of the present invention 1 dental pulp mescenchymal stem cell is frozen is apparently higher than standard
Cell viability after frozen stock solution (matched group) is frozen.
The above is only the preferred embodiment of the present invention it is noted that ordinary skill people for 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 (10)
1. a kind of frozen stock solution of dental pulp mescenchymal stem cell is it is characterised in that include DMSO, FBS and dental pulp mescenchymal stem cell
Conditioned medium；
The preparation method of the conditioned medium of described dental pulp mescenchymal stem cell is：Dental pulp mescenchymal stem cell adopts cell culture
Base carries out cell culture, treats that cell confluency degree reaches 80%～90%, collects cell culture medium supernatant, obtains dental pulp mesenchyme
The conditioned medium of stem cell.
2. frozen stock solution according to claim 1 is it is characterised in that described DMSO, described FBS are done with described dental pulp mesenchyme
The volume ratio of the conditioned medium of cell is (1～3)：(3～5)：(4～6).
3. frozen stock solution according to claim 1 is it is characterised in that described DMSO, described FBS are done with described dental pulp mesenchyme
The volume ratio of the conditioned medium of cell is 1：4：5.
4. frozen stock solution according to claim 1 is it is characterised in that described cell culture medium is the DMEM/ containing 10%FBS
F12 culture medium.
5. frozen stock solution according to claim 1 is it is characterised in that the dental pulp mescenchymal stem cell of described cell culture is inoculated
Density is (5～10) × 103/cm2.
6. frozen stock solution according to any one of claim 1 to 5 is it is characterised in that described collection cell culture medium supernatant
The step being centrifuged also is included after liquid.
7. frozen stock solution according to claim 6 is it is characterised in that the step that also includes filtration sterilization after described centrifugation.
8. a kind of cryopreservation methods of dental pulp mescenchymal stem cell are it is characterised in that adopt any one of claim 1 to 7
Frozen stock solution frozen dental pulp mescenchymal stem cell.
9. cryopreservation methods according to claim 8 are it is characterised in that in terms of mL/cell, frozen stock solution is done with dental pulp mesenchyme
The ratio of cell is 1：(1～2) × 106.
10. cryopreservation methods according to claim 8 are it is characterised in that described frozen temperature≤- 80 DEG C.
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| CN107252477A (en) * | 2017-06-13 | 2017-10-17 | 广州赛莱拉干细胞科技股份有限公司 | A kind of stem cell medicine and its preparation method and application |
| CN109938010A (en) * | 2019-03-20 | 2019-06-28 | 江苏瑞思坦生物科技有限公司 | A kind of human adipose mesenchymal stem cells transport liquid and preparation method thereof |
| 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 |
| CN112029712A (en) * | 2019-05-14 | 2020-12-04 | 北京泰生生物科技有限公司 | Separation and culture method and application of gingival stem cells |
| 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 |
| CN112998008A (en) * | 2021-03-04 | 2021-06-22 | 山东博森医学工程技术有限公司 | Dental pulp stem cell cryopreservation protective solution and dental pulp stem cell cryopreservation method |
| CN115044544A (en) * | 2022-02-22 | 2022-09-13 | 张建秋 | Recovery method of dental pulp mesenchymal stem cells |
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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 |
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| CN115044544A (en) * | 2022-02-22 | 2022-09-13 | 张建秋 | Recovery method of dental pulp mesenchymal stem cells |
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