CN104771414A - Adipose-derived stem cell preparation and preparation method thereof - Google Patents
Adipose-derived stem cell preparation and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the field of biology and medicines, and especially relates to an adipose-derived stem cell preparation and a preparation method thereof. The adipose-derived stem cell preparation comprises adipose-derived stem cells, hyaluronic acid, alhumin and normal saline. The adipose-derived stem cells can maintain good survival rate and activity, and do not differentiate under the protection of hyaluronic acid, alhumin and normal saline in the adipose-derived stem cell preparation. The preparation method of the adipose-derived stem cell preparation provided by the invention can avoid damages of the adipose-derived stem cells, and improves the survival rate of the adipose-derived stem cells in the preparation. Experiments show that the adipose-derived stem cell preparation can maintain the survival rate of the adipose-derived stem cells above 99%, and allows the cells to have uniform size and shuttle-shaped form. After the adipose-derived stem cells are preserved at 0-4DEG C for 72h, the cells still maintain good form and have no volume enlargement or other abnormal phenomena, the survival rate still maintains above 95%, and the adipose-derived stem cells still maintain the characteristics of stem cells, and do not differentiate.
Description
Technical field
The present invention relates to biomedicine field, particularly relate to a kind of fat stem cell preparation and preparation method thereof.
Background technology
Osteoarthritis (OA) is machinery and biological factor interaction, makes the chronic disabling disease of articular chondrocytes, extracellular matrix generation pathological changes; Mostly occur at middle-aged and elderly people, along with the increase prevalence at age obviously raises.Current Therapeutic Method comprises operation and pharmaceutical intervention, but the normal cartilage function of patient is still difficult to recover.Therefore, cartilage injury's reparation is the thorny problem in clinical research always.In recent years, people are using the repair cell of mesenchymal stem cells MSCs as osteoarthritis.But want to obtain bone marrow stem cell, just have to pass through puncture bone marrow extraction, and then carry out separation and purification extraction.In addition, bone marrow stem cell content is low, oncogenicity is high, and application risk is self-evident clinically.Fat stem cell abundance, easily obtain, oncogenicity is low, and has powerful propagation and differentiation potential, become the cartilage replacement therapy study hotspot of very attractive.Fat stem cell (Adipose-derived stem cells, ADSCs) is from fatty tissue, be separated a kind of stem cell with multi-lineage potential obtained in recent years; Have can increase rapidly, the not easily feature such as old and feeble.Research finds, fat stem cell, except being differentiated to form new cartilaginous tissue, can also secrete the various active factor, as: transforminggrowthfactor-β1, Keratiocyte growth element etc.Result of study in recent years shows, fat stem cell can be used in treating joint disease.
But fat stem cell is very fragile, is made into preparation and is difficult to ensure survival rate, if the survival rate deficiency of fat stem cell in preparation, good drug effect can not be played.Therefore, improve further fat stem cell survival rate in the formulation and activity, ensure that the stem cell transplanted survives very necessary in vivo.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is fat stem cell preparation providing a kind of stem cell survival high and preparation method thereof.
Fat stem cell preparation provided by the invention, comprising: fat stem cell, hyaluronic acid, albumin and normal saline.
In certain embodiments, in stem cell medicine provided by the invention, hyaluronic mass fraction is 3% ~ 5%.
As preferably, hyaluronic mass fraction is 3%.
In certain embodiments, in stem cell medicine provided by the invention, albuminous mass fraction is 5% ~ 10%.
As preferably, albuminous mass fraction is 10%.
In certain embodiments, in stem cell medicine provided by the invention, the density of fat stem cell is 2 × 10
7individual/mL ~ 5 × 10
7individual/mL.
As preferably, the density of fat stem cell is 3 × 10
7individual/mL.
Preferably, in stem cell medicine provided by the invention, hyaluronic mass fraction is 3%, albuminous mass fraction is 10%, and the density of fat stem cell is 3 × 10
7individual/mL.
Preferably, in stem cell medicine provided by the invention, hyaluronic mass fraction is 4%, albuminous mass fraction is 5%, and the density of fat stem cell is 2 × 10
7individual/mL.
Preferably, in stem cell medicine provided by the invention, hyaluronic mass fraction is 5%, albuminous mass fraction is 10%, and the density of fat stem cell is 5 × 10
7individual/mL.
In preparation, stem cells density is excessive, and survival rate significantly can reduce because of undernutrition, and the too small maintenance being also unfavorable for survival rate of stem cells density, and too small stem cells density can cause the increase by required amount of formulation.The present invention confirms by experiment, and stem cells density is 2 × 10
7individual/mL ~ 5 × 10
7individual/mL time, stem cell survival is the highest.
Normal saline refers to Physiology Experiment or the osmotic pressure conventional clinically sodium chloride solution equal with the osmotic pressure of animal or human's body blood plasma.The normal saline that the present invention adopts refers to that mass fraction is the sodium chloride solution of 0.9%, and it is consistent with the osmotic pressure of tissue, using it as solvent, can not produce damage to fat stem cell.
Albumin (also known as albumin, albumin, Alb) is the protein that in blood plasma, content is maximum, accounts for 40% ~ 60% of Total plasma protein.The constant of plasma colloid osmotic pressure can be maintained and bear certain transportation function.Show in research in the past, albumin may the differentiation of induced dry-cell.But the present invention confirms by experiment, in fat stem cell preparation, add albumin can provide nutrition for fat stem cell, improve fat stem cell survival rate in the formulation, and fat stem cell preparation has not yet to see the sign of differentiation in low-temperature preservation after one month.Within the specific limits, albuminous addition becomes positive correlation with the survival rate of fat stem cell.But too high albumin concentration can cause cell dehydration distortion because of osmotic unbalances, reduces the survival rate of fat stem cell on the contrary.Experiment shows, add in preparation albuminous mass fraction be 5% ~ 10% be conducive to most improve cell survival rate.
Hyaluronic acid, also known as hyaluronic acid, is a kind of high molecular polymer.The higher polysaccharides be made up of unit D-Glucose aldehydic acid and N-acetyl-glucosamine.Hyaluronic acid is the main component forming the connective tissues such as human body cell interstitial, vitreum, knuckle synovia, the important physiological function playing water conservation in vivo, maintain extracellular space, regulate osmotic pressure, lubrication, promotion cytothesis.According to the size of molecular weight, hyaluronic acid can be divided into: macromolecule hyaluronic acid, middle-molecular-weihydroxyethyl hyaluronic acid, small-molecular-weight hyaluronic acid three class.Wherein: the hyaluronic molecular weight of macromolecule is 1800000 ~ 2200000, be mainly used in preventing dehydration, histiocyte regenerates, and compact skin; The hyaluronic molecular weight of middle-molecular-weihydroxyethyl is 1000000 ~ 1800000, is mainly used in the skin that compacts, permanent moisturizing; The hyaluronic molecular weight of small-molecular-weight is 400000 ~ 1000000, is mainly used in fast and cell generation hydration, keeps the moisture of cell.In fat stem cell preparation, add hyaluronic acid can provide suitable matrix environment for fat stem cell, maintain the survival rate of fat stem cell, and be beneficial to fat stem cell in the process for the treatment of joint disease, be divided into cartilaginous tissue.
In certain embodiments, in stem cell medicine provided by the invention, hyaluronic molecular weight is 400000 ~ 1000000.
In certain embodiments, in stem cell medicine provided by the invention, fat stem cell is the fat stem cell in the third generation ~ the 5th generation.
The differentiation potential that the fat stem cell in the third generation ~ the 5th generation can keep stem cell intrinsic, can not go out the phenomenon of apoptosis or decline, and its state is best, under suitable condition, can increase rapidly and break up.
As preferably, the fat stem cell in the third generation ~ the 5th generation cleans with normal saline after pancreatin enzymolysis.
By the fat stem cell in the third generation ~ the 5th generation after pancreatin enzymolysis, be used further to normal saline cleaning the survival rate that fat stem cell preparation provided by the invention can ensure stem cell further.
Wherein, pancreatin enzymolysis is specially: the fat stem cell getting the third generation ~ the 5th generation (P3-P5), after PBS cleaning twice, adds 2 ~ 3mL 0.25% pancreatin-0.02%EDTA; 80% fat stem cell shrinkage becomes bowlder, adds the DMEM culture medium termination enzymolysis of 5 ~ 8mL containing 10%FBS, repeatedly blows and beats cell, until cell all comes off with liquid-transfering gun.
Wherein, cleaning is specially: the fat stem cell suspension through pancreatin enzymolysis is transferred in 50mL centrifuge tube, the centrifugal 3 ~ 5min of 500 ~ 800g.Centrifugal end abandons supernatant afterwards, adds the normal saline re-suspended cell of 20 ~ 40mL in cell precipitation, and the centrifugal 3 ~ 5min of 500 ~ 800g, abandons supernatant.
Preferably, the number of times of cleaning is 2 times.
The preparation method of fat stem cell preparation provided by the invention, comprising: after albumin, hyaluronic acid being mixed with normal saline, resuspended fat stem cell, obtains fat stem cell preparation.
The preparation method of fat stem cell preparation provided by the invention is simply gentle, can not the activity of damaged stem cells, thus improves the survival rate of stem cell in fat stem cell preparation.
Concrete, the preparation method of fat stem cell preparation provided by the invention comprises: hyaluronic acid and normal saline being mixed to hyaluronic final concentration is after 3% ~ 5%, and resuspended fat stem cell is 2 × 10 to the density of fat stem cell
7individual/mL ~ 5 × 10
7individual/mL.
Fat stem cell preparation provided by the invention can be good at the activity and the survival rate that maintain fat stem cell, during preservation can not break up, and therefore, fat stem cell preparation provided by the invention may be used for the medicine preparing treatment joint disease.
The application of fat stem cell preparation provided by the invention in the medicine of preparation treatment joint disease.
As preferably, joint disease is osteoarthritis.
Fat stem cell preparation provided by the invention, comprising: fat stem cell, hyaluronic acid, albumin and normal saline.In fat stem cell preparation provided by the invention, fat stem cell, under the protection of hyaluronic acid and albumin and normal saline, can maintain good survival rate and activity, and can not start differentiation.And the preparation method of fat stem cell preparation provided by the invention, can avoid damaging fat stem cell, improve the survival rate of fat stem cell in preparation.Experiment shows, the survival rate of fat stem cell can be maintained more than 99% by fat stem cell preparation provided by the invention, and cell size is even, form is shuttle-type.After 0 DEG C ~ 4 DEG C preservation 72h, cell still remains good form, does not occur that volume becomes large and waits abnormal phenomena, survival rate still remains on more than 95%, and detects through surface antigen, and fat stem cell still keeps the feature of stem cell, do not break up, there is good activity.
Accompanying drawing explanation
Fig. 1 shows the three ~ five fat subsitutes stem cell morphology figure, and wherein, Fig. 1-a shows that third generation fat stem cell amplifies 40 times; Fig. 1-b shows that third generation fat stem cell amplifies 100 times; Fig. 1-c shows that the 5th fat subsitutes stem cell amplifies 40 times; Fig. 1-d shows that the 5th fat subsitutes stem cell amplifies 100 times;
Fig. 2 shows fat stem cell volume size distribution figure;
Fig. 3 shows that the Trypan Blue result in 3h prepared by fat stem cell preparation that the embodiment of the present invention 4 provides;
Fig. 4 shows the testing result of the fat stem cell preparation that the embodiment of the present invention 2 provides, and wherein, Fig. 4-a shows the Trypan Blue result of fat stem cell; Fig. 4-b shows the testing result of flow cytomery fat stem cell surface antigen HLA-DR; Fig. 4-c shows the testing result of flow cytomery fat stem cell surface antigen CD45;
Fig. 5 shows the testing result of the fat stem cell preparation that the embodiment of the present invention 3 provides, and wherein, Fig. 5-a shows the Trypan Blue result of fat stem cell; Fig. 5-b shows the testing result of flow cytomery fat stem cell surface antigen HLA-DR; Fig. 5-c shows the testing result of flow cytomery fat stem cell surface antigen CD45;
Fig. 6 shows the testing result of the fat stem cell preparation that the embodiment of the present invention 4 provides, and wherein, Fig. 6-a shows the Trypan Blue result of fat stem cell; Fig. 6-b shows the testing result of flow cytomery fat stem cell surface antigen HLA-DR; Fig. 6-c shows the testing result of flow cytomery fat stem cell surface antigen CD45;
Fig. 7 shows the testing result of the fat stem cell preparation that comparative example 1 of the present invention provides, and wherein, Fig. 7-a shows the Trypan Blue result of fat stem cell; Fig. 7-b shows the testing result of flow cytomery fat stem cell surface antigen HLA-DR; Fig. 7-c shows the testing result of flow cytomery fat stem cell surface antigen CD45;
Fig. 8 shows the testing result of the fat stem cell preparation that comparative example 2 of the present invention provides, and wherein, Fig. 8-a shows the Trypan Blue result of fat stem cell; Fig. 8-b shows the testing result of flow cytomery fat stem cell surface antigen HLA-DR; Fig. 8-c shows the testing result of flow cytomery fat stem cell surface antigen CD45;
Fig. 9 shows the testing result of the fat stem cell preparation that comparative example 3 of the present invention provides, and wherein, Fig. 9-a shows the Trypan Blue result of fat stem cell; Fig. 9-b shows the testing result of flow cytomery fat stem cell surface antigen HLA-DR; Fig. 9-c shows the testing result of flow cytomery fat stem cell surface antigen CD45;
Figure 10 shows the testing result of the bone marrow stem cell preparation that comparative example 4 of the present invention provides, and wherein, Figure 10-a shows the testing result of flow cytomery fat stem cell surface antigen FSC; Figure 10-b shows the testing result of flow cytomery fat stem cell surface antigen HLA-DR; Figure 10-c shows the testing result of flow cytomery fat stem cell surface antigen CD45.
Detailed description of the invention
The invention provides a kind of fat stem cell preparation and preparation method thereof, 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 and application are described by preferred embodiment, related personnel obviously can change methods and applications herein or suitably changes and combination not departing from content of the present invention, spirit and scope, realizes and applies the technology of the present invention.
The instrument that the present invention adopts is all common commercially available product, all can buy in market.
Below in conjunction with embodiment, set forth the present invention further:
The preparation of embodiment 1 fat stem cell
1) be separated
Body adipose tissue divides and is filled in 50mL centrifuge tube, often pipe 20mL.Often add isopyknic 0.5% NTx enzyme (final concentration is 0.25%) in pipe, fully mix, sealing, is transferred in Tempeerature-constant air shaking table, 37 DEG C, 100R digests 1h.Often pipe adds the FBS of 4mL, stops digestion, mixing.Centrifugal, the centrifugal 5min of 1500rpm/min.Discard two-layer liquid, often to add 40mL PBS resuspended for pipe, re-suspended cell.Centrifugal, the centrifugal 5min of 1500rpm/min, supernatant discarded, obtain fat stem cell, this stem cell is seed.
2) cultivate
Add the DMEM-F12 culture medium re-suspended cell containing 15%FBS, with 1 × 10
5individual/mL inoculation.Rock back and forth culture dish gently, at the bottom of cell suspension is uniformly distributed in bottle; Transfer to 37 DEG C, 5%CO
2, saturated humidity is cultivate in the incubator of 95%.Change liquid first after 24h, discard suspension cell. within every 3 days subsequently, change a not good liquor.Fat stem cell is observed under inverted microscope, if cell fusion reaches 85%, then the fat stem cell in fat stem cell is gone down to posterity process, 3rd ~ 5 generations of acquisition.(as shown in Figure 1) observe and detect cell size.(as shown in Figure 2)
As shown in the figure: fat stem cell P3 and P5 for time, cell size is evenly, form is shuttle-type, merge to 85% time present swirling.Fat stem cell division growth speed is fast, in good condition.Known from fat stem cell aspect graph, fat stem cell, between 16-22um, meets fat stem cell volume size distribution, describes fat stem cell and does not occur that volume becomes large and waits abnormal phenomena.
3) enzymolysis
Get the fat stem cell of the third generation to the 5th generation (P3-P5), after PBS cleaning twice, add 2 ~ 3mL 0.25% pancreatin-0.02%EDTA; Observe under being placed in inverted microscope, 80% fat stem cell shrinkage becomes bowlder, adds the DMEM culture medium termination enzymolysis of 5 ~ 8mL containing 10%FBS, repeatedly blows and beats cell, until cell all comes off, obtain the fat stem cell after enzymolysis with liquid-transfering gun.
4) clean
Fat stem cell suspension after enzymolysis is transferred in 50mL centrifuge tube, the centrifugal 3 ~ 5min of 500 ~ 800g.Centrifugal end abandons supernatant afterwards, adds the normal saline re-suspended cell of 20 ~ 40mL in cell precipitation, the centrifugal 3 ~ 5min of 500 ~ 800g, abandons supernatant (this step repeats once).Fat stem cell after cleaning is used for the preparation of preparation.
Embodiment 2 ~ 4
Albumin and small-molecular-weight hyaluronic acid are joined in normal saline, obtains the substrate of preparation, the fat stem cell after the cleaning prepared by the resuspended embodiment 1 of substrate.In each embodiment, the amount of albumin, hyaluronic acid, fat stem cell is as shown in table 1:
Table 1 embodiment 2 ~ 4
| Albumin mass fraction | Hyaluronic acid mass fraction | Fat stem cell number | |
| Embodiment 2 | 5％ | 4％ | 2×10 7Individual/mL |
| Embodiment 3 | 10％ | 5％ | 5×10 7Individual/mL |
| Embodiment 4 | 10％ | 3％ | 3×10 7Individual/mL |
Comparative example 1 ~ 3
Albumin and small-molecular-weight hyaluronic acid are joined in normal saline, obtains the substrate of preparation, the fat stem cell after the cleaning prepared by the resuspended embodiment 1 of substrate.In each embodiment, the amount of albumin, hyaluronic acid, fat stem cell is as shown in table 2:
Table 2 comparative example 1 ~ 3
| Albumin mass fraction | Hyaluronic acid mass fraction | Fat stem cell number | |
| Comparative example 1 | 0％ | 4％ | 2×10 7Individual/mL |
| Comparative example 2 | 2.5％ | 5％ | 5×10 7Individual/mL |
| Comparative example 3 | 20％ | 3％ | 3×10 7Individual/mL |
Comparative example 4 ~ 6
Albumin and small-molecular-weight hyaluronic acid are joined in normal saline, obtains the substrate of preparation, with the resuspended bone marrow stem cell of substrate.In each embodiment, the amount of albumin, hyaluronic acid, bone marrow stem cell is as shown in table 3:
Table 3 comparative example 4 ~ 6
| Albumin mass fraction | Hyaluronic acid mass fraction | Fat stem cell number | |
| Embodiment 2 | 5％ | 4％ | 2×10 7Individual/mL |
| Embodiment 3 | 10％ | 5％ | 5×10 7Individual/mL |
| Embodiment 4 | 10％ | 3％ | 3×10 7Individual/mL |
The evaluation of embodiment 5 quality of the pharmaceutical preparations
Quality Identification is carried out to the stem cell medicine that embodiment 2 ~ 4 and comparative example 1 ~ 6 obtain.Each sample checks 3 times.Method is:
After preparation is made, detect fresh obtained preparation in 3h, Detection of content comprises: the content of antibacterial etc. in stem cell morphology, survival rate, preparation.Wherein, cellular morphology detection, cell viability detect and adopt trypan blue staining.Testing result is as shown in table 4.Fig. 3 shows that the Trypan Blue result in 3h prepared by fat stem cell preparation that the embodiment of the present invention 4 provides; In the fat stem cell preparation that other embodiments are obtained, the form of stem cell similarly.
Testing result in rear 3h made by table 4 preparation
Note: A represent cell size evenly, in shuttle-type
B represents that cell volume increases, occurs the abnormal morphologies such as oval or irregular
* represent that there is significant difference p<0.05
--show and do not examine
Result shows: in preparation 3h, in fat stem cell preparation provided by the invention, stem cell size is even, and survival rate is high, and under trypan blue detects, cell all refuses dye (dead cell is dyed to blueness, and living cells refuses dye); Describe fat stem cell and do not occur death, vigor is good.And in stem cell medicine prepared by comparative example 1 ~ 6, the survival rate of stem cell is lower slightly, and what have has even occurred abnormal morphology.
After preparation is made, load in syringe, after 0 DEG C ~ 4 DEG C preservation 72h, again detect stem cell morphology, survival rate, surface antigen, the content of antibacterial etc. in preparation.Wherein, cellular morphology detects, cell viability detects and adopts trypan blue staining, and surface antigen detects and adopts flow cytometry analysis.Testing result is as shown in table 5 and Fig. 4 ~ 10.
Testing result after rear 72h made by table 5 preparation
Note: A represent cell size evenly, in shuttle-type
B represents that cell volume increases, occurs the abnormal morphologies such as oval or irregular
* represent that there is significant difference p<0.05
--show and do not examine
Associative list 4 ~ 5 and Fig. 4 ~ 10, result shows, after preparation 72h, in fat stem cell preparation prepared by embodiment 2 ~ 4, stem cell morphology is little and homogeneous, survival rate remarkable (p<0.05) is higher than comparative example 1 ~ 6, refractivity is strong and from the result of flow cytometer showed, fat stem cell high expressed CD59 and CD90, and expression rate is respectively and is greater than 99%; Low expression of HLA-DR and CD45 are less than 1%.Meet the surface antigen feature of stem cell, describe the phenomenon that differentiation does not appear in fat stem cell, still maintain the feature of stem cell.Also have no the appearance differentiating phenomenon of stem cell in comparative example 1 ~ 3, but in comparative example 4 ~ 6 there is differentiating phenomenon in stem cell.
In addition, the stem cell medicine that provides of embodiment 2 ~ 4 and comparative example 1 ~ 6 is in the detections of antibacterial, fungus, endotoxin and five large viruses after testing, is all negative.Describe the up-to-standard of fat stem cell preparation, can be used for the re-injection in body, carry out the treatment of osteoarthritis disorders.
Embodiment 6 preparation Clinical evaluation
Choose 24 rabbits, get 8 at random for negative control group, 16 modelings form the model of osteoarthritis.After modeling, 16 rabbits are divided into two groups at random, are respectively experimental group and positive controls.
The fat stem cell preparation that experimental group injection embodiment 4 provides, the pharmaceutical base of positive controls and positive controls injection embodiment 4 (comprises normal saline, albumin and hyaluronic acid, wherein albuminous mass fraction is 10%, and hyaluronic mass fraction is 3%).
Injecting method is:
A. percutaneous puncture is injected to osteoarthritis bone cavity.
B. percutaneous puncture osteoarthritis district Local Multipoint injection.
C. Postoperative Intravenous instillation low molecular dextran 300-600ml, continuous 3-5d; After injection first, within the 2nd week, carrying out second time, for the third time interventional therapy, is for 3 times a course for the treatment of.After injection first, within the 2nd week, the 1st month, the 2nd month, the 3rd month, evaluate.
As shown in table 6 to the testing result of cartilage thickness:
Table 6 MRI evaluates cartilage thickness (x ± S, n=3, mm)
| Number of elements | 2 weeks | 1 month | 2 months | 3 months | |
| Negative control group | 8 | 1.05±0.06 | 1.05±0.04 | 1.048±0.03 | 1.049±0.03 |
| Positive controls | 8 | 0.54±0.11* | 0.58±0.07* | 0.64±0.08* | 0.73±0.12* |
| Experimental group | 8 | 0.60±0.45* | 0.68±0.07* | 0.84±0.06* | 0.98±0.06 |
Note: * is P<0.05, has significant difference.
As shown in Table 6, there is not large change in the cartilage thickness of negative control group in three months, and cartilage thickness maintains a stable level, and thickness does not occur that diversity reduces.And positive controls, there is certain self-regeneration mechanism in it, cartilage thickness increased to some extent in 3 months, but its thickness thickens speed slowly, can not play obvious improvement result.And experimental group is after re-injection preparation, its cartilage thickness is after the reparation of 3 months, and its cartilage thickness and matched group significant difference, describe fat stem cell and can be used for treating osteoarthritis, and therapeutic effect is good.
As shown in table 7 to Mankin histological scores result:
Table 7 each treatment group pathology Mankin scoring is compared (x ± S)
| Number of elements | 2 weeks | 1 month | 2 months | 3 months | |
| Negative control group | 8 | 1.325±0.22 | 1.26±0.15 | 4.06±0.17* | 1.24±0.31 |
| Positive controls | 8 | 5.41±0.47 | 5.34±0.62 | 5.26±0.55* | 5.10±0.46* |
| Experimental group | 8 | 4.50±0.45* | 4.06±0.17* | 3.53±0.16* | 2.66±0.18* |
Note: * is P<0.05, has significant difference.
As shown in Table 7, there is not large change in the chondropathology Mankin scoring of negative control group, cartilaginous tissue maintains a stable level, does not occur that pathologic changes in three months.And positive controls, there is certain self-regeneration mechanism in it, chondropathology Mankin score value declined to some extent in 3 months, but it improves speed slowly, can not play obvious improvement result.And experimental group is after re-injection preparation, its cartilage thickness is after the reparation of 3 months, although its chondropathology Mankin score value and matched group exist significant difference, but still plays obvious improvement result.
Below be only the preferred embodiment of the present invention; 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 make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a fat stem cell preparation, is characterized in that, comprising: fat stem cell, hyaluronic acid, albumin and normal saline.
2. fat stem cell preparation according to claim 1, is characterized in that, wherein said albuminous mass fraction is 5% ~ 10%.
3. fat stem cell preparation according to claim 1, is characterized in that, wherein said hyaluronic mass fraction is 3% ~ 5%.
4. fat stem cell preparation according to claim 1, is characterized in that, wherein hyaluronic molecular weight is 400000 ~ 1000000.
5. fat stem cell preparation according to claim 1, is characterized in that, the density of wherein said fat stem cell is 2 × 10
7individual/mL ~ 5 × 10
7individual/mL.
6. fat stem cell preparation according to claim 1, is characterized in that, the density of wherein said fat stem cell is 3 × 10
7individual/mL.
7. fat stem cell preparation according to claim 1, is characterized in that, wherein said fat stem cell is the fat stem cell in the third generation ~ the 5th generation.
8. fat stem cell preparation according to claim 4, is characterized in that, the fat stem cell in the described third generation ~ the 5th generation cleans with normal saline after pancreatin enzymolysis.
9. the preparation method of the fat stem cell preparation described in any one of claim 1 ~ 8, is characterized in that, comprising: after albumin, hyaluronic acid being mixed with normal saline, resuspended fat stem cell, obtains fat stem cell preparation.
10. the application of the fat stem cell preparation as described in any one of claim 1 ~ 8 in the medicine of preparation treatment joint disease.
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| CN106729642A (en) * | 2017-03-17 | 2017-05-31 | 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 |
| CN107603945A (en) * | 2016-11-24 | 2018-01-19 | 广东万海细胞生物科技有限公司 | A kind of preparation method for the new culture for improving autologous fat stem cell propagation |
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| WO2021219003A1 (en) * | 2020-04-30 | 2021-11-04 | 中国医学科学院北京协和医院 | Adipose-derived stem cell preparation, preparation method therefor and use thereof |
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| CN105168251A (en) * | 2015-09-09 | 2015-12-23 | 广州赛莱拉干细胞科技股份有限公司 | Stem cell preparation as well as preparation method and application thereof |
| CN107603945A (en) * | 2016-11-24 | 2018-01-19 | 广东万海细胞生物科技有限公司 | A kind of preparation method for the new culture for improving autologous fat stem cell propagation |
| CN106729642A (en) * | 2017-03-17 | 2017-05-31 | 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 |
| CN112237589A (en) * | 2019-07-19 | 2021-01-19 | 丰泽康生物医药（深圳）有限公司 | Umbilical cord mesenchymal stem cell active matter and preparation method and application thereof |
| WO2021219003A1 (en) * | 2020-04-30 | 2021-11-04 | 中国医学科学院北京协和医院 | Adipose-derived stem cell preparation, preparation method therefor and use thereof |
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