CN105238749A - Method for resuscitating mesenchymal stem cells - Google Patents
Method for resuscitating mesenchymal stem cells Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and discloses a method for resuscitating mesenchymal stem cells. The method comprises the following steps: culturing the mesenchymal stem cells which go down to the generations P3 to P5 by utilizing a complete culture medium, then culturing the mesenchymal stem cells by utilizing a serum-free DMEM culture medium, culturing the mesenchymal stem cells respectively for 24h, 48h and 72h, then collecting culture medium supernatant at each time period, centrifuging the supernatant at each time period, then collecting the supernatants, mixing the supernatants to obtain a condition culture medium; and unfreezing the frozen mesenchymal stem cells, adding the unfrozen mesenchymal stem cells into the condition culture medium, centrifuging to remove the supernatant, then adding the condition culture medium again, re-suspending, and culturing until the fusion degree of the cell meets the requirement. The culture medium supernatant culturing the mesenchymal stem cells is used as a culture medium for resuscitation and is used for substituting the commercial culture medium such as alpha-MEM and DMEM generally adopted in the prior art, the growth of the resuscitated mesenchymal stem cells is promoted by virtue of active substances secreted by various cells during the multiplication process, and the activity is improved.
Description
Technical field
The present invention relates to biological technical field, relate to a kind of method of recovery mesenchymal stem cells MSCs specifically.
Background technology
Mescenchymal stem cell (mesenchymalstemcells, MSC) be the important member of stem cell line, derive from and grow early stage mesoderm and ectoderm, belong to multipotential stem cell, MSC finds at first in marrow, because of its there is multi-lineage potential, the feature such as hematopoiesis support and promotion stem cell are implanted, immunoregulation and self-replacation and day by day receive the concern of people.If mescenchymal stem cell is in vivo or under external specific inductive condition, the Various Tissues cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium can be divided into, still there is multi-lineage potential after continuous passage cultivation and freezen protective, can be used as the injuries of tissues and organs reparation that desirable seed cell causes for old and feeble and pathology.
Mesenchymal stem cells MSCs is the mescenchymal stem cell deriving from adult bone marrow, and the same with umbilical cord, fat mesenchymal stem cell, it has stronger self and multi-lineage potential.Mescenchymal stem cell needs to be preserved by cryogenic freezing after separation and Culture, carries out injuries of tissues and organs reparation more in use through recovery.
Document " after cryopreservation resuscitation mesenchymal stem cells MSCs amplification in vitro and multi-lineage potential research " describes the common method of current recovery mesenchymal stem cells MSCs (journal of Zhejiang university engineering version, the 40th volume o. 11th, in November, 2006):
From liquid nitrogen, take out sample put into 37 DEG C of water-baths immediately shake cryopreservation tube when rewarming, after 1-2min, rewarming is complete, add fresh nutrient solution (being the α-MEM nutrient solution of the FBS of 10% containing volume fraction) the centrifugal 5min of 400g, abandon supernatant liquor, add fresh medium and put into CO
2cultivate in incubator.Reach after 80%-90% fusion until cell, go down to posterity with Digestive system digestion and with 1: 3 and increase further.
Although the Measures compare of above-mentioned recovery is simple, quick, the cell viability after the method recovery is on the low side, is unfavorable for follow-up carrying out the clinical applications such as injuries of tissues and organs reparation.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of method of recovery mesenchymal stem cells MSCs, make described method can improve the vigor of the rear mesenchymal stem cells MSCs of recovery, and still keep the ability that can be divided into scleroblast, stearoblast.
To achieve these goals, the invention provides following technical scheme:
A method for recovery mesenchymal stem cells MSCs, comprising:
After step 1, the mesenchymal stem cells MSCs that will be passaged to P3-P5 generation are first cultivated with mescenchymal stem cell perfect medium, use the DMEM culture medium culturing of serum-free again, substratum supernatant is collected respectively when cultivating 24h, 48h, 72h, after centrifugal for each time period supernatant, get supernatant mixing again, obtain conditioned medium;
Step 2, frozen mesenchymal stem cells MSCs to be thawed, and join and centrifugally in the conditioned medium that step 1 collects remove supernatant, and then it is resuspended and be cultured to cytogamy degree and reach requirement to add conditioned medium.
To recover the not high problem of cell viability after frozen mesenchymal stem cells MSCs for existing method, the present invention does not adopt commercial medium usual in traditional method to recover when recovery, but adopt the conditioned medium cultivating mesenchymal stem cells MSCs to recover, significantly can improve the phenomenon that the rear cell viability of recovery is not high.The present invention finds to utilize conditioned medium effectively can improve the vigor of recovery cell or tissue, and the vigor of the conditioned medium of different sources to different cell, tissue has different promotions or restraining effect.
Wherein, as preferably, step 1 is:
To the mesenchymal stem cells MSCs in P3-P5 generation be passaged to according to 5000-10000/cm
2cell density with mescenchymal stem cell perfect medium cultivate 24h, after Hanks cleaning, use the DMEM culture medium culturing of serum-free again, substratum supernatant is collected respectively when cultivating 24h, 48h, 72h, the centrifugal 5-10min of 1000-2000rpm, remove precipitation, collect the mixing of all supernatants, after membrane filtration, obtain conditioned medium.
The parameter of the cell-seeding-density in conditioned medium of the present invention, centrifugally operated, incubation time and environment etc. can be regulated according to practical situation, the present invention is only the parameter providing comparative optimization, but be not limited in these parameters and combination thereof, provided preferred parameter and combination thereof are also not limited only to for the similar parameters occurred in additive method step of the present invention.
As preferably, described mescenchymal stem cell perfect medium is the DMEM/F12 substratum containing 10%FBS.
Mesenchymal stem cells MSCs for P3-P5 generation can obtain according to the separation method of this area routine and propagating method, the invention provides following preferred preparation method:
Mononuclear cell layer is isolated from Normal Human Bone Marrow sample, eccentric cleaning cell precipitation, cultivate 48h by the resuspended precipitation of growth of marrow mesenchyme stem cell substratum, then change growth of marrow mesenchyme stem cell substratum and continue to be cultured to cytogamy degree and reach 80%-90%, obtain P0 generation;
With tryptic digestion P0 for attached cell, centrifugal rear growth of marrow mesenchyme stem cell substratum re-suspended cell precipitation, and Secondary Culture, be passaged to P3-P5 generation in the manner described.
Wherein, further preferably, described mononuclear cell layer can be separated by following methods and obtain:
The sample of bone marrow getting normal people adds the mixing of equal-volume physiological saline, and then draw marrow mixed solution with pasteur pipet and join in isopyknic lymphocyte separation medium, centrifugal rear pasteur pipet draws the mononuclear cell layer in middle layer.
Further preferred, described growth of marrow mesenchyme stem cell substratum is the DMEM substratum containing 10%FBS and 10ng/mLbFGF.
In step 2 recovery link, conditioned medium of the present invention is substituted the commercial medium of existing frequent employing, recover according to general way.
As preferably, step 2 is:
Frozen mesenchymal stem cells MSCs is placed in the water-bath of 35-42 DEG C, shake incubation 30-60 second, then join in the conditioned medium of 5-10 times of volume, with the centrifugal 3-5 minute of 1000-2000rpm, remove supernatant, by the resuspended precipitation of conditioned medium, piping and druming mixing, and according to 5000-10000/cm
2cell density conditioned medium be placed on 37 DEG C, 5%CO
2incubator quiescent culture is after 24 hours, and replacing conditioned medium continues to be cultured to cytogamy degree and reaches 80%-90%.
The method of the invention is used to recover frozen mesenchymal stem cells MSCs, compare the cell after adopting existing method (method for resuscitation recorded in " after cryopreservation resuscitation mesenchymal stem cells MSCs amplification in vitro and multi-lineage potential research ") recovery, its vigor significantly improves, and still keep the biological property that mescenchymal stem cell is good, there is the ability being divided into scleroblast and stearoblast.Based on this, the invention provides the application of described conditioned medium in recovery mesenchymal stem cells MSCs.Conditioned medium of the present invention can be placed on-20 DEG C of incubators and preserve 6 months about-12 months or be placed on-80 DEG C of preservations more than 1 year when not using.When needing to use, return to 37 DEG C of normal temperature and use.
From above technical scheme, the present invention was to cultivate the substratum supernatant of mesenchymal stem cells MSCs for substratum during recovery, substitute the such as α-MEM, this commercial medium of DMEM that generally adopt in existing method, the active substance relying on the various kinds of cell wherein comprised to secrete in breeding promotes the growth of the mesenchymal stem cells MSCs after recovering, and then improves its vigor.
Accompanying drawing explanation
Figure 1 shows that vigor column diagram after method for resuscitation of the present invention and existing method for resuscitation cell recovery;
Figure 2 shows that method for resuscitation of the present invention and existing method for resuscitation cell recovery and total cellular score column diagram after cultivating 72h;
Figure 3 shows that blank group cell-surface antigens flow cytomery figure;
Figure 4 shows that experimental group cell-surface antigens flow cytomery figure;
Figure 5 shows that the microscopy figure of the rear cell osteogenic induction of recovery;
Figure 6 shows that the microscopy figure of the rear cell adipogenic induction of recovery.
Embodiment
The embodiment of the invention discloses a kind of method of recovery mesenchymal stem cells MSCs.Those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention is described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope product as herein described and method are changed or suitably change with combination, realize and apply the technology of the present invention.
In order to understand the present invention further, be described in detail below in conjunction with the method for embodiment to a kind of recovery mesenchymal stem cells MSCs provided by the invention.
Embodiment 1: the cultivation of mesenchymal stem cells MSCs and the collection of conditioned medium
By the adult bone marrow extracted, with adding isopyknic physiological saline, then drawing marrow mixed solution with pasteur pipet and joining in isopyknic lymphocyte separation medium, leaving the heart 10 minutes with 2000; Centrifugal rear pasteur pipet draws the mononuclear cell layer in middle layer.Add growth of marrow mesenchyme stem cell substratum (DMEM+10%FBS+10ng/mlbFGF) re-suspended cell precipitation, be placed on 37 DEG C subsequently, 5%CO
2cell culture incubator is cultivated, and every 2-3 days changes liquid, treats that cytogamy degree reaches 80%-90%, uses 0.25% trypsin digestion cell, the resuspended precipitation of centrifugal rear perfect medium, 5 × 10
3/ cm
2continue in cell density inoculation culture ware to cultivate.Get P3-P5 and carry out conditioned medium collection for cell.
Conditioned medium is collected: for the mesenchymal stem cells MSCs of conditioned medium, first according to 5 × 10
3/ cm
2cell density inoculation culture ware, perfect medium carried out cultivation after 24 hours, after Hanks cleaning, add the DMEM substratum of serum-free, after 24 hours, 48 hours, 72 hours, collecting cell substratum supernatant respectively, supernatant is sub-packed in 50mL centrifuge tube, the centrifugal 5min of 1000pm.Remove precipitation, collect supernatant.Draw conditioned medium with disposable syringe, with 0.22 μm of membrane filtration, be collected in sterile collection bottle.Be placed on-20 DEG C of incubators and preserve 6 months about-12 months or be placed on-80 DEG C of preservations more than 1 year.When needing to use, return to normal temperature and use.
Embodiment 2: the recovery of mesenchymal stem cells MSCs
Frozen mesenchymal stem cells MSCs is taken out from liquid nitrogen, be placed in the water-bath of 35-42 DEG C, concussion incubation 60 seconds, after cell thawing, immediately frozen storing liquid to be joined in the conditioned medium of 5 times of volumes cell suspension with 1000rpm centrifugal 5 minutes, remove supernatant, by the resuspended precipitation of conditioned medium; Blow and beat mixing gently, and according to 7 × 10
3/ cm
2in cell density inoculation culture ware, cultivate with conditioned medium.Be placed on 37 DEG C, 5%CO
2incubator left standstill after 24 hours, changed conditioned medium and continued to cultivate, and observation of cell amplification every day situation, cell carries out continuing to be cultured to cytogamy degree and reaches 80%-90%.
Embodiment 3: recovery cell viability contrasts
Experimental group: prepare conditioned medium according to embodiment 1, frozen mesenchymal stem cells MSCs is taken out from liquid nitrogen, is placed in the water-bath of 35-42 DEG C, concussion incubation 60 seconds, after cell thawing, joined by frozen storing liquid in the conditioned medium of 5 times of volumes immediately, take a morsel cell suspension, adds trypan blue, be placed on cell viability calculating instrument and carry out cell viability detection, all the other cell suspensions centrifugal 5 minutes with 1000rpm, remove supernatant, by the resuspended precipitation of conditioned medium; Blow and beat mixing gently, and according to 7 × 10
3/ cm
2cell density inoculation diameter is in 15cm culture dish, cultivates with conditioned medium.Be placed on 37 DEG C, 5%CO
2incubator cultivates 72 hours, uses trysinization eccentric cell, uses conditioned medium re-suspended cell, and take a morsel cell suspension, adds trypan blue, is placed on cell counter and carries out cell counting.
Control group: take out mesenchymal stem cells MSCs (be the mesenchymal stem cells MSCs in identical source with experimental group) and put into 37 DEG C of water-baths immediately and shake cryopreservation tube from liquid nitrogen, after 1-2min, rewarming is complete, add fresh nutrient solution (volume fraction is the α-MEM nutrient solution of the FBS of 10%), take a morsel cell suspension, add trypan blue, be placed on cell viability calculating instrument and carry out cell viability detection, subsequently with the centrifugal 5min of 400g, abandon supernatant liquor, add fresh medium, according to 7 × 10
3/ cm
2cell density inoculation diameter is in 15cm culture dish, puts into 37 DEG C, 5%CO
2to cultivate in incubator. cell carries out cultivation 72 hours, use trysinization eccentric cell, with fresh nutrient solution (volume fraction is the α-MEM nutrient solution of the FBS of 10%), take a morsel cell suspension, add trypan blue, be placed on cell counter and carry out cell counting.
Both see Fig. 1 and Fig. 2 by comparing result, can obviously be found out by Fig. 1, and the vigor of the mesenchymal stem cells MSCs after recovering via the inventive method is close to 95%, and the cell viability after the recovery of existing method does not reach 90%, and both compare has significant difference.Meanwhile, as seen from Figure 2, at cell recovery and after the cultivation of 72h, through the inventive method cultured cells sum considerably beyond existing method, show that the cell viability after method for resuscitation recovery of the present invention is higher, multiplication capacity is strong.
Embodiment 4: the mesenchymal stem cells MSCs surface marker after flow cytomery recovery
According to embodiment 1,2 recovery culturing cell, after recovering, cytogamy degree reaches 80%-90%, with 0.25% trysinization eccentric cell, and counting after often pipe add 2 × 10
5cell count, dye solution washes 1 time, the centrifugal 5min of 1000rpm; Abandon supernatant, with dye solution piping and druming mixing cell; Add each 2 μ L of CD73, CD90, CD45 and HLA-DRA antibody, and set a pipe as blank; At 4 DEG C, lucifuge reaction 15-20min; Dye solution is washed once, the centrifugal 5min of 1000rpm; The cell of direct mark abandons supernatant, and lucifuge adds the sample-loading buffer of 500 μ L, and mixing, with 200 eye mesh screen filtration cell samples, flow cytomery cell-surface antigens, the results are shown in Figure 3 and Fig. 4.
From Fig. 3 and Fig. 4, the negative surface marker CD45 of mescenchymal stem cell (white corpuscle is positive), HLA-DR (MHC-II quasi-molecule) are for all to present feminine gender, and mescenchymal stem cell surface marker CD73, CD90 all presents the positive simultaneously.After recovery is described, mesenchymal stem cells MSCs still keeps the good biological feature of mescenchymal stem cell.
Embodiment 5: the mescenchymal stem cell induced osteogenesis differentiation qualification of recovery derived from bone marrow
According to embodiment 1,2 recovery culturing cell, after recovering, cytogamy degree reaches 80%-90%, with 0.25% trysinization eccentric cell, according to 1 × 10
3cell/cm
2be inoculated in six orifice plates, add perfect medium, after 24h, add after Osteogenic Induction Medium carries out cultivation 24h, changed liquid every 2-3 days, carry out Alizarin red staining after 21 days, the results are shown in Figure 5.
By the visible calcium scoring of Alizarin red staining, mescenchymal stem cell after recovery, through osteogenic induction after 3 weeks, illustrates that the mesenchymal stem cells MSCs of recovering through conditioned medium still keeps the potential to Osteoblast Differentiation.
Embodiment 6: the mescenchymal stem cell of recovery derived from bone marrow induces into fat differentiation qualification
According to embodiment 1,2 recovery culturing cell, after recovering, cell confluency degree reaches 80%-90%, with 0.25% trysinization eccentric cell, after collecting cell, and according to 1 × 10
3cell/cm
2be inoculated in six orifice plates, add perfect medium, after 24h, add adipogenic induction substratum and cultivate.Adipogenic induction substratum comprises basic medium DMEM, 10% foetal calf serum, 0.5mMIBMX, 1 μM of dexamethasone, 100 μMs of indomethacins, 5 μ g/mL Regular Insulin, 2mm/L glutamine etc.Within every three days, change liquid.Carry out oil red O stain after surrounding, qualification fat drips formational situation, the results are shown in Figure 6.
Mesenchymal stem cells MSCs after recovery, through adipogenic induction after 4 weeks, by oil red O stain red color visible oil droplet, illustrates that the mesenchymal stem cells MSCs of recovering through conditioned medium still keeps the potential to becoming fat differentiation.
The explanation of above embodiment just understands method of the present invention and core concept thereof for helping.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also fall in the protection domain of the claims in the present invention.
Claims (8)
1. a method for recovery mesenchymal stem cells MSCs, is characterized in that, comprising:
After step 1, the mesenchymal stem cells MSCs that will be passaged to P3-P5 generation are first cultivated with mescenchymal stem cell perfect medium, use the DMEM culture medium culturing of serum-free again, substratum supernatant is collected respectively when cultivating 24h, 48h, 72h, after centrifugal for each time period supernatant, get supernatant mixing again, obtain conditioned medium;
Step 2, frozen mesenchymal stem cells MSCs to be thawed, and join and centrifugally in the conditioned medium that step 1 collects remove supernatant, and then it is resuspended and be cultured to cytogamy degree and reach requirement to add conditioned medium.
2. method according to claim 1, it is characterized in that, step 1 is:
To the mesenchymal stem cells MSCs in P3-P5 generation be passaged to according to 5000-10000/cm
2cell density with mescenchymal stem cell perfect medium cultivate 24h, after Hanks cleaning, use the DMEM culture medium culturing of serum-free again, substratum supernatant is collected respectively when cultivating 24h, 48h, 72h, the centrifugal 5-10min of 1000-2000rpm, remove precipitation, collect the mixing of all supernatants, after membrane filtration, obtain conditioned medium.
3. method according to claim 1 or 2, is characterized in that, described P3-P5 obtains by the following method for mesenchymal stem cells MSCs:
Mononuclear cell layer is isolated from Normal Human Bone Marrow sample, eccentric cleaning cell precipitation, cultivate 48h by the resuspended precipitation of growth of marrow mesenchyme stem cell substratum, then change growth of marrow mesenchyme stem cell substratum and continue to be cultured to cytogamy degree and reach 80%-90%, obtain P0 generation;
With tryptic digestion P0 for attached cell, centrifugal rear growth of marrow mesenchyme stem cell substratum re-suspended cell precipitation, and Secondary Culture, be passaged to P3-P5 generation in the manner described.
4. method according to claim 3, is characterized in that, described mononuclear cell layer can be separated by following methods and obtain:
The sample of bone marrow getting normal people adds the mixing of equal-volume physiological saline, and then draw marrow mixed solution with pasteur pipet and join in isopyknic lymphocyte separation medium, centrifugal rear pasteur pipet draws the mononuclear cell layer in middle layer.
5. method according to claim 3, is characterized in that, described growth of marrow mesenchyme stem cell substratum is the DMEM substratum containing 10%FBS and 10ng/mLbFGF.
6. method according to claim 1 or 2, is characterized in that, described mescenchymal stem cell perfect medium is the DMEM/F12 substratum containing 10%FBS.
7. method according to claim 1, it is characterized in that, step 2 is:
Frozen mesenchymal stem cells MSCs is placed in the water-bath of 35-42 DEG C, shake incubation 30-60 second, then join in the conditioned medium of 5-10 times of volume, with the centrifugal 3-5 minute of 1000-2000rpm, remove supernatant, by the resuspended precipitation of conditioned medium, piping and druming mixing, and according to 5000-10000/cm
2cell density conditioned medium be placed on 37 DEG C, 5%CO
2incubator quiescent culture is after 24 hours, and replacing conditioned medium continues to be cultured to cytogamy degree and reaches 80%-90%.
8. the application of conditioned medium described in claim 1 in recovery mesenchymal stem cells MSCs.
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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 |
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