CN105219707A - A kind of method of recovery fat mesenchymal stem cell - Google Patents
A kind of method of recovery fat mesenchymal stem cell Download PDFInfo
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- CN105219707A CN105219707A CN201510800480.4A CN201510800480A CN105219707A CN 105219707 A CN105219707 A CN 105219707A CN 201510800480 A CN201510800480 A CN 201510800480A CN 105219707 A CN105219707 A CN 105219707A
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Abstract
The present invention relates to biological technical field, disclose a kind of method of recovery fat mesenchymal stem cell.The method will be passaged to the fat mesenchymal stem cell in P3-P5 generation with after complete culture medium culturing, use the DMEM/F12 culture medium culturing of serum-free again, collecting substratum supernatant when cultivating 24h, 48h, 72h respectively, after centrifugal for each supernatant, get supernatant mixing again, obtain conditioned medium; Frozen fat mesenchymal stem cell is thawed, and joins and centrifugally in conditioned medium remove supernatant, and then it is resuspended and be cultured to cytogamy degree and reach requirement to add conditioned medium.The present invention was to cultivate the substratum supernatant of fat mesenchymal stem cell for substratum during recovery, substitute the mescenchymal stem cell perfect medium generally adopted in existing method, the active substance relying on the various kinds of cell wherein comprised to secrete in breeding promotes the growth of the fat mesenchymal stem cell after recovering, and then improves its vigor.
Description
Technical field
The present invention relates to biological technical field, relate to a kind of method of recovery fat mesenchymal stem cell 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.
Fat mesenchymal stem cell is the mescenchymal stem cell deriving from adult adipose, and the same with umbilical cord, mesenchymal stem cells MSCs, 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 " separation and Culture of human adipose mesenchymal stem cells and frozen front and back biology change " (Chinese neuroimmunology and neurological magazine in July, 2,013, the 20th volume the 4th phase) describes the common method of current recovery fat mesenchymal stem cell:
From liquid nitrogen, take out cell after 6 months be placed in the centrifuge tube that is transferred to after 38 DEG C of-40 DEG C of water-baths melt rapidly and nutrient solution is housed in advance immediately with the centrifugal 5min of 300g, be transferred to cellar culture in culturing bottle with ADSCs perfect medium re-suspended cell, ADSCs perfect medium is the low sugar DMEM containing 10% volume fraction foetal calf serum.
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 fat mesenchymal stem cell, make described method can improve the vigor of the rear fat mesenchymal stem cell 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 fat mesenchymal stem cell, comprising:
After step 1, the fat mesenchymal stem cell that will be passaged to P3-P5 generation are first cultivated with mescenchymal stem cell perfect medium, use the DMEM/F12 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 fat mesenchymal stem cell 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 fat mesenchymal stem cell for existing method, the present invention does not adopt commercial medium (mescenchymal stem cell perfect medium) usual in traditional method to recover when recovery, but adopt the conditioned medium cultivating fat mesenchymal stem cell 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 fat mesenchymal stem cell 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/F12 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.
Fat mesenchymal stem cell 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:
By the adult adipose 0.1%-0.5%I Collagenase Type enzymolysis, digestion extracted, centrifuged deposit mescenchymal stem cell perfect medium is resuspended and cultivate, and every 2-3 days changes liquid, treats that cytogamy degree reaches 80%-90%, obtains P0 generation;
With trysinization P0 for cell, the resuspended precipitation of centrifugal rear mescenchymal stem cell perfect medium Secondary Culture, be passaged to P3-P5 generation in the manner described.
Further preferred, by the adult adipose extracted, after PBS cleaning, shred fatty tissue, and add 0.1%-0.5%I Collagenase Type, at 37 DEG C, under 100-200rpm condition, digest enzymolysis 30-50 minute, leave heart 5-10 minute with 1000-2000, remove supernatant, add mescenchymal stem cell perfect medium re-suspended cell precipitation, be placed on 37 DEG C subsequently, 5%CO
2cell cultures is cultivated, and every 2-3 days changes liquid, treats that cytogamy degree reaches 80%-90%, obtains P0 generation;
With 0.25% trysinization P0 for cell, the resuspended precipitation of centrifugal rear mescenchymal stem cell perfect medium, 5000-10000/cm
2continue in cell density inoculation culture ware to cultivate, be passaged to P3-P5 generation in the manner described.
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 fat mesenchymal stem cell 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 fat mesenchymal stem cell, compare the cell after adopting existing method (method for resuscitation recorded in " separation and Culture of human adipose mesenchymal stem cells and the change of frozen front and back biology ") 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 fat mesenchymal stem cell.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 fat mesenchymal stem cell for substratum during recovery, substitute the mescenchymal stem cell perfect medium generally adopted in existing method, the active substance relying on the various kinds of cell wherein comprised to secrete in breeding promotes the growth of the fat mesenchymal stem cell 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 fat mesenchymal stem cell.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 fat mesenchymal stem cell provided by the invention.
Embodiment 1: the cultivation of fat mesenchymal stem cell and the collection of conditioned medium
By the adult adipose extracted, after PBS cleaning, shred fatty tissue, and add 0.25I Collagenase Type, at 37 DEG C, enzymolysis is digested 50 minutes under 200rpm condition, leave heart 5-10 minute with 1000-2000, remove supernatant, add perfect medium (DMEM/F12+10%FBS) re-suspended cell precipitation, be placed on 37 DEG C subsequently, 5%CO
2cell cultures 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) × 10
3continue in/cm2 cell density inoculation culture ware to cultivate.Get P3-P5 and carry out conditioned medium collection for cell.
Conditioned medium is collected: for the fat mesenchymal stem cell of conditioned medium, first according to (5-10) × 10
3/ cm
2cell density inoculation culture ware, perfect medium carried out cultivation after 24 hours, after Hanks cleaning, add the DMEM/F12 substratum of serum-free, after 24 hours, 48 hours, 72 hours, collecting cell substratum supernatant, is sub-packed in 50mL centrifuge tube, the centrifugal 5-10min of 1000-2000rpm by supernatant respectively.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 fat mesenchymal stem cell
Frozen fat mesenchymal stem cell 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 fat mesenchymal stem cell 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 is inoculated in six orifice plates, 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 viability calculating instrument and carries out cell viability detection.
Control group: take out fat mesenchymal stem cell (be the fat mesenchymal stem cell in identical source with experimental group) from liquid nitrogen, drops in 38 DEG C of-40 DEG C of water-baths immediately and melts.Be transferred in the centrifuge tube be equipped with in advance containing the DMEM/F12 nutrient solution of 10%FBS, take out cell after mixing cell and carry out cell viability detection, with the centrifugal 5min of 300g, remove supernatant, with hADSCs perfect medium re-suspended cell, according to 7 × 10
3/ cm
2cell density inoculates six orifice plates, and is positioned over 37 DEG C, 5%CO
2cultivate 72 hours in incubator.Use trysinization eccentric cell, with the DMEM/F12 substratum re-suspended cell containing 10%FBS, take a morsel cell suspension, adds trypan blue, is placed on cell counter and carries 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 fat mesenchymal stem cell after recovering via the inventive method is about 92%, and the cell viability after the recovery of existing method is about 85%, 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 fat mesenchymal stem cell 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, fat mesenchymal stem cell still keeps the good biological feature of mescenchymal stem cell.
Embodiment 5: the mescenchymal stem cell induced osteogenesis differentiation qualification of recovering adipose-derived
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 fat mesenchymal stem cell of recovering through conditioned medium still keeps the potential to Osteoblast Differentiation.
Embodiment 6: adipose-derived mescenchymal stem cell of recovering 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.
Fat mesenchymal stem cell after recovery, through adipogenic induction after 4 weeks, by oil red O stain red color visible oil droplet, illustrates that the fat mesenchymal stem cell 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 (7)
1. a method for recovery fat mesenchymal stem cell, is characterized in that, comprising:
After step 1, the fat mesenchymal stem cell that will be passaged to P3-P5 generation are first cultivated with mescenchymal stem cell perfect medium, use the DMEM/F12 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 fat mesenchymal stem cell 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 fat mesenchymal stem cell 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/F12 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 fat subsitutes mescenchymal stem cell obtains by the following method:
By the adult adipose 0.1%-0.5%I Collagenase Type enzymolysis, digestion extracted, centrifuged deposit mescenchymal stem cell perfect medium is resuspended and cultivate, and every 2-3 days changes liquid, treats that cytogamy degree reaches 80%-90%, obtains P0 generation;
With trysinization P0 for cell, the resuspended precipitation of centrifugal rear mescenchymal stem cell perfect medium Secondary Culture, be passaged to P3-P5 generation in the manner described.
4. method according to claim 3, it is characterized in that, described P3-P5 fat subsitutes mescenchymal stem cell obtains by the following method:
By the adult adipose extracted, after PBS cleaning, shred fatty tissue, and add 0.1%-0.5%I Collagenase Type, at 37 DEG C, enzymolysis 30-50 minute is digested under 100-200rpm condition, leave heart 5-10 minute with 1000-2000, remove supernatant, add mescenchymal stem cell perfect medium re-suspended cell precipitation, be placed on 37 DEG C subsequently, 5%CO
2cell cultures is cultivated, and every 2-3 days changes liquid, treats that cytogamy degree reaches 80%-90%, obtains P0 generation;
With 0.25% trysinization P0 for cell, the resuspended precipitation of centrifugal rear mescenchymal stem cell perfect medium, 5000-10000/cm
2continue in cell density inoculation culture ware to cultivate, be passaged to P3-P5 generation in the manner described.
5. 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.
6. method according to claim 1, it is characterized in that, step 2 is:
Frozen fat mesenchymal stem cell 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%.
7. the application of conditioned medium described in claim 1 in recovery fat mesenchymal stem cell.
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| CN111849880B (en) * | 2020-06-23 | 2022-04-29 | 和携科技有限公司 | Recovery method of human adipose mesenchymal stem cells after ultralow-temperature cryopreservation |
| CN112155008A (en) * | 2020-09-30 | 2021-01-01 | 北京银丰鼎诚生物工程技术有限公司 | Cryopreservation and recovery method of adipose tissues and adipose tissue cryopreservation recovery kit |
| CN115305232A (en) * | 2022-02-22 | 2022-11-08 | 徐营奎 | Adipose-derived mesenchymal stem cell recovery culture solution and recovery method |
| CN115305232B (en) * | 2022-02-22 | 2024-02-20 | 王泰华 | Adipose-derived mesenchymal stem cell resuscitating culture solution and resuscitating method |
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