CN105238749B - A kind of method of recovery mesenchymal stem cell - Google Patents
A kind of method of recovery mesenchymal stem cell Download PDFInfo
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- CN105238749B CN105238749B CN201510791413.0A CN201510791413A CN105238749B CN 105238749 B CN105238749 B CN 105238749B CN 201510791413 A CN201510791413 A CN 201510791413A CN 105238749 B CN105238749 B CN 105238749B
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Abstract
The present invention relates to field of biotechnology, disclose a kind of method of recovery mesenchymal stem cell.This method will be passaged to the mesenchymal stem cell in P3-P5 generation first with after complete medium culture, the DMEM culture medium culture of serum-free is used again, respectively culture for 24 hours, 48h, 72h when collect culture medium supernatant, will each period supernatant centrifugation after take supernatant to mix again, obtain conditioned medium；The mesenchymal stem cell frozen is thawed, and is added in conditioned medium centrifugation and removes supernatant, conditioned medium is then added again and is resuspended and cultivates to cell fusion degree reaches requirement.The present invention was to cultivate culture medium of the culture medium supernatant of mesenchymal stem cell as recovery when, such as α-MEM, this commercial medium of DMEM generallyd use in substitution existing method, the growth of mesenchymal stem cell after promoting recovery by the active material that various kinds of cell wherein included is secreted in breeding, and then improve vigor.
Description
Technical field
The present invention relates to field of biotechnology, a kind of method for particularly relating to recovery mesenchymal stem cell.
Background technique
Mescenchymal stem cell (mesenchymal stem cells, MSC) is the important member of stem cell line, is derived from
The mesoderm and ectoderm of mesoderm growing early stage belong to multipotential stem cell, and MSC initially has found in marrow, because it is with Multidirectional Differentiation
Potential, hematopoiesis support and promote stem cell implantation, immunoregulation and the concern that people are increasingly subject to the features such as self-replacation.Such as
Mescenchymal stem cell is in vivo or in vitro under specific inductive condition, can be divided into fat, bone, cartilage, muscle, tendon, ligament,
The Various Tissues cell such as nerve, liver, cardiac muscle, endothelium still has multi-lineage potential after continuous passage culture and freezen protective, can
As ideal seed cell for injuries of tissues and organs reparation caused by aging and lesion.
Mesenchymal stem cell is derived from the mescenchymal stem cell of adult bone marrow, dry thin with umbilical cord, fat mesenchymal
Born of the same parents are the same, with stronger self-renewing and multi-lineage potential.Mescenchymal stem cell needs after being separately cultured by low
Warm freezen protective carries out injuries of tissues and organs reparation using recovery when in use.
Document " mesenchymal stem cell amplification in vitro and multi-lineage potential research after cryopreservation resuscitation " (learn by Zhejiang University
Report engineering version, the o. 11th of volume 40, in November, 2006) describe the common method of current recovery mesenchymal stem cell:
It it is removed from liquid nitrogen sample in rewarming is immediately placed in 37 DEG C of water-baths and shake cryopreservation tube, rewarming is complete after 1-2min
Finish, fresh culture solution (α-MEM culture solution containing the FBS that volume fraction is 10%) 400g be added and is centrifuged 5min, abandons supernatant,
Fresh medium is added and is put into CO2It is cultivated in incubator.After cell reaches 80%-90% fusion, digested with digestive juice and with 1
: 3 passages further expand
Although the method for above-mentioned recovery is fairly simple, quick, the cell viability after this method recovery is relatively low, is unfavorable for
The clinical applications such as subsequent progress injuries of tissues and organs reparation.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of methods of recovery mesenchymal stem cell, so that described
Method can be improved the vigor of mesenchymal stem cell after recovery, and still maintains and can be divided into osteoblast, at rouge
The ability of cell.
To achieve the goals above, the invention provides the following technical scheme:
A kind of method of recovery mesenchymal stem cell, comprising:
The mesenchymal stem cell for being passaged to P3-P5 generation is first used mescenchymal stem cell complete medium culture by step 1
Afterwards, then with the DMEM culture medium culture of serum-free, respectively culture for 24 hours, 48h, 72h when collect culture medium supernatant, by each time
It takes supernatant to mix again after Duan Shangqing centrifugation, obtains conditioned medium；
Step 2, by the mesenchymal stem cell frozen thaw, and be added to step 1 collection conditioned medium in from
The heart removes supernatant, and conditioned medium is then added again and is resuspended and cultivates to cell fusion degree and reach requirement.
The not high problem of cell viability after the mesenchymal stem cell frozen for existing method recovery, the present invention is multiple
It does not use commercial medium usual in conventional method to recover when Soviet Union, but uses and cultivated mesenchymal stem cell
Conditioned medium is recovered, and the not high phenomenon of cell viability after recovery can be significantly improved.Present invention discover that being trained using condition
Nutrient solution can effectively improve the vigor of recovery cell or tissue, and the conditioned medium of separate sources is to different cells, tissue
Vigor has different promotion or inhibiting effect.
Wherein, preferably, step 1 are as follows:
The mesenchymal stem cell in P3-P5 generation will be passaged to according to 5000-10000/cm2Cell density mesenchyma
The culture of stem cell complete medium for 24 hours, after being cleaned with Hanks, then with the DMEM culture medium culture of serum-free, is being cultivated respectively
For 24 hours, culture medium supernatant is collected when 48h, 72h, 1000-2000rpm is centrifuged 5-10min, and it is mixed to collect all supernatants for removal precipitating
It closes, obtains conditioned medium after membrane filtration.
For parameter, incubation time and the ring of cell-seeding-density, centrifugally operated in conditioned medium of the present invention
Border etc. can be adjusted according to the actual situation, and the present invention is provided than more preferably parameter, it is not limited to these parameters
And combinations thereof, for the similar parameters occurred in other methods step of the present invention be also not limited to provided by preferred parameter and its
Combination.
Preferably, the mescenchymal stem cell complete medium is the DMEM/F12 culture medium containing 10%FBS.
The mesenchymal stem cell in P3-P5 generation can be come according to the separation method and propagating method of this field routine
It obtains, the present invention provides following preferred preparation methods:
Mononuclear cell layer is isolated from Normal Human Bone Marrow sample, eccentric cleaning cell precipitation is dry thin with medulla mesenchyma
Intracellular growth culture medium, which is resuspended, to be precipitated and cultivates 48h, is then replaced growth of marrow mesenchyme stem cell culture medium and is continued to cultivate to thin
Born of the same parents' degrees of fusion reaches 80%-90%, obtains P0 generation；
With trypsin digestion P0 for attached cell, cell is resuspended with growth of marrow mesenchyme stem cell culture medium after centrifugation
Precipitating, and secondary culture, are passaged to P3-P5 generation in the manner described.
Wherein, still more preferably, the mononuclear cell layer can be separated by following methods and be obtained:
It takes the sample of bone marrow of normal person that isometric physiological saline is added to mix, then draws marrow mixed liquor with pasteur pipet
It is added in isometric lymphocyte separation medium, draws the mononuclear cell layer of middle layer after centrifugation with pasteur pipet.
Still more preferably, the growth of marrow mesenchyme stem cell culture medium is containing 10%FBS and 10ng/mL
The DMEM culture medium of bFGF.
In step 2 recovery link, by conditioned medium of the present invention substitute it is existing through frequently with business culture
Base is recovered according to general way.
Preferably, step 2 are as follows:
The mesenchymal stem cell frozen is placed in 35-42 DEG C of water-bath, concussion incubation 30-60 seconds, then
It is added in the conditioned medium of 5-10 times of volume, with 1000-2000rpm centrifugation 3-5 minutes, removes supernatant, use conditioned medium
Precipitating is resuspended, piping and druming mixes, and according to 5000-10000/cm2Cell density be placed on 37 DEG C, 5%CO with conditioned medium2
After incubator stationary culture 24 hours, replacement conditioned medium continues culture to cell fusion degree and reaches 80%-90%.
The mesenchymal stem cell frozen with the method for the invention recovery (" is frozen multiple compared to using existing method
After Soviet Union mesenchymal stem cell amplification in vitro and multi-lineage potential research " in record method for resuscitation) recovery after cell,
Its vigor significantly improves, and still keep the good biological property of mescenchymal stem cell, have be divided into osteoblast and at
The ability of lipocyte.Based on this, the present invention provides application of the conditioned medium in recovery mesenchymal stem cell.
Conditioned medium of the present invention can be placed in -20 DEG C of incubators when not in use and save -12 months 6 months or so or place
Saved 1 year at -80 DEG C or more.It needs to use in use, being restored to 37 DEG C of room temperature.
From the above technical scheme, when the present invention was to cultivate the culture medium supernatant of mesenchymal stem cell as recovery
Culture medium, substitute such as α-MEM, this commercial medium of DMEM that generally use in existing method, dependence is wherein included
The active material that various kinds of cell is secreted in breeding come promote recovery after mesenchymal stem cell growth, Jin Erti
Its high vigor.
Detailed description of the invention
Fig. 1 show vigor column diagram after method for resuscitation of the present invention and existing method for resuscitation cell recovery；
Fig. 2 show method for resuscitation of the present invention and existing method for resuscitation cell recovery and cultivates the total number of cells column after 72h
Shape figure；
Fig. 3 show blank group cell surface antigen flow cytomery figure；
Fig. 4 show experimental group cell surface antigen flow cytomery figure；
Fig. 5 show the microscopy figure of cell osteogenic induction after recovery；
Fig. 6 show the microscopy figure of cell adipogenic induction after recovery.
Specific embodiment
The embodiment of the invention discloses a kind of methods of recovery mesenchymal stem cell.Those skilled in the art can borrow
Reflect present disclosure, is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to this field
It is it will be apparent that they are considered as being included in the present invention for technical staff.Method of the invention passes through preferable reality
Example is applied to be described, related personnel obviously can not depart from the content of present invention, in spirit and scope to product as described herein
And method is modified or appropriate changes and combinations, carrys out implementation and application the technology of the present invention.
It is dry to a kind of recovery medulla mesenchyma provided by the invention below with reference to embodiment for a further understanding of the present invention
The method of cell is described in detail.
Embodiment 1: the culture of mesenchymal stem cell and the collection of conditioned medium
Then the adult bone marrow of extraction is drawn into marrow mixed liquor with pasteur pipet with isometric physiological saline is added
It is added in isometric lymphocyte separation medium, is centrifuged 10 minutes with 2000 turns；Middle layer is drawn with pasteur pipet after centrifugation
Mononuclear cell layer.Growth of marrow mesenchyme stem cell culture medium (DMEM+10%FBS+10ng/ml bFGF) is added to be resuspended carefully
Born of the same parents' precipitating, is subsequently placed at 37 DEG C, 5%CO2Cell incubator culture, changes liquid in every 2-3 days, reaches 80%- to cell fusion degree
90%, with 0.25% trypsin digestion cell, it is resuspended and is precipitated with complete medium after centrifugation, 5 × 103/cm2Cell density inoculation training
It supports and continues to cultivate in ware.P3-P5 is taken to carry out conditioned medium collection for cell.
Conditioned medium is collected: for the mesenchymal stem cell of conditioned medium, first according to 5 × 103/cm2Cell is close
It spends inoculated and cultured ware and after being cleaned with Hanks, the DMEM culture medium of serum-free is added after complete medium carries out culture 24 hours,
After 24 hours, 48 hours, 72 hours, cell culture medium supernatant is collected respectively, supernatant is sub-packed in 50mL centrifuge tube,
1000pm is centrifuged 5min.Removal precipitating, collects supernatant.Conditioned medium is drawn with disposable syringe, with 0.22 μm of filter membrane mistake
Filter is collected in sterile collection bottle.It is placed on -20 DEG C of incubators and saves -12 months 6 months or so or be placed on -80 DEG C of preservations 1
Year or more.It needs in use, being restored to room temperature use.
Embodiment 2: the recovery of mesenchymal stem cell
The mesenchymal stem cell frozen is removed from liquid nitrogen, is placed in 35-42 DEG C of water-bath, concussion incubates
60 seconds, after cell thaws, frozen stock solution is added to in the conditioned medium of 5 times of volumes cell suspension immediately with 1000rpm centrifugation 5
Minute, supernatant is removed, is resuspended and is precipitated with conditioned medium；Gently piping and druming mixes, and according to 7 × 103/cm2Cell density inoculated and cultured
In ware, with conditioned medium culture.37 DEG C are placed on, 5%CO2After incubator stands 24 hours, replacement conditioned medium continues
Culture, daily to observe cell amplification situation, cell carries out continuing to cultivate to cell fusion degree reaching 80%-90%.
Embodiment 3: recovery cell viability comparison
Experimental group: conditioned medium is prepared according to embodiment 1, by the mesenchymal stem cell frozen from liquid nitrogen
It takes out, is placed in 35-42 DEG C of water-bath, concussion incubates 60 seconds, and after cell thaws, frozen stock solution is added to 5 times of bodies immediately
In long-pending conditioned medium, a small amount of cell suspension is taken, trypan blue is added, cell viability calculating instrument is placed on and carries out cell viability inspection
It surveys, remaining cell suspension removes supernatant with 1000rpm centrifugation 5 minutes, is resuspended and is precipitated with conditioned medium；Gently piping and druming mixes, and
According to 7 × 103/cm2It is in 15cm culture dish, with conditioned medium culture that cell density, which is inoculated with diameter,.It is placed on 37 DEG C, 5%
CO2Incubator culture 72 hours, centrifuge cell is digested with pancreatin, cell is resuspended with conditioned medium, takes a small amount of cell suspension, adds
Enter trypan blue, is placed on cell counter and carries out cell count.
Control group: it is (dry for the medulla mesenchyma of identical source with experimental group to be removed from liquid nitrogen mesenchymal stem cell
Cell) it is immediately placed in 37 DEG C of water-baths and shakes cryopreservation tube, rewarming finishes after 1-2min, and fresh culture solution (volume point is added
Number for 10% FBS α-MEM culture solution), take a small amount of cell suspension, trypan blue be added, be placed on cell viability calculating instrument into
The detection of row cell viability, is then centrifuged 5min with 400g, abandons supernatant, fresh medium is added, according to 7 × 103/cm2Cell is close
Degree inoculation diameter is to be put into 37 DEG C, 5%CO in 15cm culture dish2Cell is cultivated in incubator and carries out culture 72 hours, uses pancreas
Enzymic digestion centrifuge cell takes a small amount of cell outstanding with fresh culture solution (α-MEM culture solution for the FBS that volume fraction is 10%)
Trypan blue is added in liquid, is placed on cell counter and carries out cell count.
The two comparing result is shown in Fig. 1 and Fig. 2, by Fig. 1, it is apparent that between marrow after recovering via the method for the present invention
The vigor of mesenchymal stem cells is close to 95%, and the cell viability after existing method recovery is not up to 90%, and the two is compared to have and be shown
Write difference.Meanwhile as seen from Figure 2, in cell recovery and after the culture of 72h, by the thin of the method for the present invention culture
Born of the same parents' sum shows that the cell viability after method for resuscitation of the present invention is recovered is higher, proliferative capacity considerably beyond existing method
By force.
Embodiment 4: the mesenchymal stem cell surface marker after flow cytomery recovery
It recovers according to embodiment 1,2 and cultivates cell, cell fusion degree reaches 80%-90% after recovering, with 0.25% pancreas
Enzymic digestion centrifuge cell, and every pipe is added 2 × 10 after counting5Cell number, dye solution are washed 1 time, and 1000rpm is centrifuged 5min；
Supernatant is abandoned, is blown and beaten with dye solution and mixes cell；Each 2 μ L of CD73, CD90, CD45 and HLA-DRA antibody is added, and sets one
Pipe is blank control；At 4 DEG C, it is protected from light 15-20min；Dye solution is washed once, and 1000rpm is centrifuged 5min；Directly mark
The cell of note abandons supernatant, is protected from light the sample-loading buffer that 500 μ L are added, and mixes, with 200 mesh screen filtration cell samples, streaming is thin
Born of the same parents' instrument detects cell surface antigen, as a result sees Fig. 3 and Fig. 4.
As can be seen from figs. 3 and 4 mescenchymal stem cell feminine gender surface marker CD45 (leucocyte is positive), HLA-DR (MHC-II
Class molecule) it is feminine gender to be presented, while the positive is presented in mescenchymal stem cell surface marker CD73, CD90.After illustrating recovery
Mesenchymal stem cell still keeps the good biological feature of mescenchymal stem cell.
Embodiment 5: the mescenchymal stem cell induced osteogenesis of recovery derived from bone marrow breaks up identification
It recovers according to embodiment 1,2 and cultivates cell, cell fusion degree reaches 80%-90% after recovering, with 0.25% pancreas
Enzymic digestion centrifuge cell, according to 1 × 103Cell/cm2Be inoculated in six orifice plates, be added complete medium, for 24 hours after, be added skeletonization
After induced medium is cultivated for 24 hours, liquid was changed every 2-3 days, Alizarin red staining is carried out after 21 days, as a result sees Fig. 5.
Mescenchymal stem cell after recovery is illustrated to pass through after osteogenic induction 3 weeks by the visible calcium scoring of Alizarin red staining
The mesenchymal stem cell for crossing conditioned medium recovery still maintains potential to Osteoblast Differentiation.
Embodiment 6: the mescenchymal stem cell induction of recovery derived from bone marrow is broken up at rouge to be identified
It recovers according to embodiment 1,2 and cultivates cell, cell confluency degree reaches 80%-90% after recovering, with 0.25% pancreas
Enzymic digestion centrifuge cell, after collecting cell, and according to 1 × 103Cell/cm2It is inoculated in six orifice plates, complete medium is added,
After for 24 hours, adipogenic induction culture medium is added and is cultivated.Adipogenic induction culture medium includes basal medium DMEM, 10% tire ox blood
Clearly, 0.5mM IBMX, 1 μM of dexamethasone, 100 μM of Indomethacins, 5 μ g/mL insulin, 2mm/L glutamine etc..It changes every three days
Liquid.Oil red O stain is carried out after four weeks, is identified fat drips formational situation, is as a result seen Fig. 6.
Mesenchymal stem cell after recovery, by oil red O stain red color visible oil droplet, is said after adipogenic induction 4 weeks
The bright mesenchymal stem cell by conditioned medium recovery is still maintained to the potential broken up at rouge.
The above description of the embodiment is only used to help understand the method for the present invention and its core ideas.It should be pointed out that pair
For those skilled in the art, without departing from the principle of the present invention, the present invention can also be carried out
Some improvements and modifications, these improvements and modifications also fall within the scope of protection of the claims of the present invention.
Claims (5)
1. a kind of method of recovery mesenchymal stem cell characterized by comprising
Step 1 will be passaged to the mesenchymal stem cell in P3-P5 generation according to 5000-10000/cm2Cell density with containing
The DMEM/F12 culture medium culture of 10%FBS for 24 hours, after being cleaned with Hanks, then with the DMEM culture medium culture of serum-free, respectively
Culture for 24 hours, 48h, 72h when collect culture medium supernatant, 1000-2000rpm be centrifuged 5-10min, removal precipitating, collect it is all on
It is clear to mix, conditioned medium is obtained after membrane filtration；
The mesenchymal stem cell frozen is placed in 35-42 DEG C of water-bath by step 2, concussion incubation 30-60 seconds, so
It is added in the conditioned medium of 5-10 times of volume afterwards, with 1000-2000rpm centrifugation 3-5 minutes, removes supernatant, use CMC model
The outstanding precipitating of base weight, piping and druming mix, and according to 5000-10000/cm2Cell density be placed on 37 DEG C, 5% with conditioned medium
CO2After incubator stationary culture 24 hours, replacement conditioned medium continues culture to cell fusion degree and reaches 80%-90%.
2. method according to claim 1, which is characterized in that the P3-P5 passes through for mesenchymal stem cell with lower section
Method obtains:
Mononuclear cell layer is isolated from Normal Human Bone Marrow sample, eccentric cleaning cell precipitation is raw with mesenchymal stem cell
Long culture medium, which is resuspended, to be precipitated and cultivates 48h, and then replacement growth of marrow mesenchyme stem cell culture medium continues to cultivate to cell and melt
It is right to reach 80%-90%, obtain P0 generation；
With trypsin digestion P0 for attached cell, cell is resuspended with growth of marrow mesenchyme stem cell culture medium after centrifugation and sinks
It forms sediment, and secondary culture, is passaged to P3-P5 generation.
3. method according to claim 2, which is characterized in that the mononuclear cell layer can be separated by following methods and be obtained:
It takes the sample of bone marrow of normal person that isometric physiological saline is added to mix, then draws marrow mixed liquor with pasteur pipet and be added
Into isometric lymphocyte separation medium, the mononuclear cell layer of middle layer is drawn after centrifugation with pasteur pipet.
4. method according to claim 2, which is characterized in that the growth of marrow mesenchyme stem cell culture medium is containing 10%
The DMEM culture medium of FBS and 10ng/mL bFGF.
5. application of the conditioned medium described in claim 1 in recovery mesenchymal stem cell.
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| CN105749347A (en) * | 2016-02-24 | 2016-07-13 | 广州赛莱拉干细胞科技股份有限公司 | Cosmetic filler and preparation method thereof |
| CN105850979A (en) * | 2016-03-14 | 2016-08-17 | 广州赛莱拉干细胞科技股份有限公司 | Cryoprotective solution and cryopreservation method for bone mesenchymal stem cells |
| JP6401818B2 (en) * | 2016-04-27 | 2018-10-10 | 株式会社金太郎ＣｅｌｌｓＰｏｗｅｒ | Method for producing activated stem cells |
| CN110129268A (en) * | 2018-02-08 | 2019-08-16 | 上海细胞治疗集团有限公司 | A kind of electricity turns the cultural method of rear PBMC |
| CN109161526A (en) * | 2018-10-12 | 2019-01-08 | 希瑞干细胞科技有限公司 | A kind of chorion mescenchymal stem cell recovery medium |
| JP6684956B1 (en) * | 2019-05-29 | 2020-04-22 | パナジー株式会社 | Cell growth method, cell growth agent and cell growth medium |
| CN110295140A (en) * | 2019-06-04 | 2019-10-01 | 河北贝特赛奥生物科技有限公司 | A kind of method of free serum culture mesenchymal stem cell |
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