CN104958320A - Cell preparation for treating osteoarthritis and preparation method thereof - Google Patents
Cell preparation for treating osteoarthritis and preparation method thereof Download PDFInfo
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Abstract
The invention provides a cell preparation for treating osteoarthritis and a preparation method thereof. The cell preparation mainly comprises mesenchymal stem cells, differentiation-induced stem cells, platelet-rich plasma and dispersion media thereof. Compared with the prior art, the cell preparation for treating osteoarthritis has the advantages that the mesenchymal stem cells are mixed with the differentiation-induced stem cells and compounded with the platelet-rich plasma and can be dispersed in the joint cavity, so that the immune regulation function of the mesenchymal stem cells can be exerted, the microenvironment of the whole joint cavity can be improved, and inflammation remission and injury repair are facilitated; tissues can be repaired after the differentiated mesenchymal stem cells are implanted into the body, so that the repair process is shortened.
Description
Technical field
The invention belongs to tissue engineering cell therapy field, be specifically related to a kind of cell preparation for the treatment of osteoarthritis and preparation method thereof.
Background technology
Because the arthritis that the age increases, the reason such as overweight, immune system abnormality, joint instability, wound causes has become the major reason affecting the normal Working Life of people and even disable.Data show, and in the crowd more than 30 years old, the prevalence of osteoarthritis is more than 6%, and this ratio improves gradually with advancing age.According to estimates, the Human Osteoarthritis of China is more than 100,000,000 people.
For the therapeutic goal that arthritis is main be at present: 1, ease the pain, swelling; 2, muscle strength, joint nutrition, blood circulation, joint lubrication is improved; 3, delay articular cartilage degeneration, retain function of joint or quality of life.
Traditional Therapeutic Method comprises expectant treatment and joint replacement.Expectant treatment generally includes physical therapy, Drug therapy etc., can short-term relief symptom, but cures the symptoms, not the disease, and the reparation of unrealized damage field, long-term effect is not good, and the state of an illness is easily repeatedly.
Joint replacement replaces pain with made joint and loses the method in the joint of function of joint, be generally applied to comparatively serious osteonecrosis, comminuted fracture dislocation can not the person of reset, pain and the osteoarthritis of moving obstacle, the rheumatoid arthritis of stiff or movable difficulty and bone tumor etc.There is prosthetic loosening in joint replacement, wear and tear or fracture, the complication such as deep infection, foreign body reaction and disincentive soft tissue calcification.
Along with the development in organizational project and regenerative medicine field, the research about tissue repair and regeneration also increases gradually.Mescenchymal stem cell is extensively present in the tissues such as bone marrow, fat, liver, peripheral blood, umbilical cord, umbilical blood, has height self-renewal capacity and multi-lineage potential, is thus widely deployed and applies.
The patent No. is the Chinese invention patent of ZL02815834.2, discloses a kind of compositions being used for the treatment of articular cartilage damage, infringement or defect.Be made up of the cellular component, culture medium and the biocompatible polymer that are separated from Cord blood, breed and/or differentiate.
Number of patent application is the Chinese invention patent application of 201010152461.2, disclose a kind of cell infusion agent being used for the treatment of bone injury and preparation method thereof, the complex that effective trophic factors of the mescenchymal stem cell of being originated by autologous bone marrow and temperature sensitive hydrogel and autoblood separation and Extraction forms.
Semi-solid or gel is formed after the biocompatible polymer of the compound recorded in above-mentioned patent or patent application document or thermosensitive hydrogel implant, be unfavorable for the dispersion of mescenchymal stem cell in articular cavity, hinder the process being improved articular cavity microenvironment by the immunoloregulation function of stem cell; In the application of cell, number of patent application is only contain mescenchymal stem cell or the single cell type of chondrocyte in the application documents of 201010152461.2, simple application mescenchymal stem cell has paracrine, immunity moderation function, but tissue repair needs to be realized by differentiation-inducing in body, it is longer therefore to treat required time; And apply merely the mescenchymal stem cell cell after induction and treat, only there is tissue repair function, do not have anti-inflammatory factors secretory function, be unfavorable for arthritic reparation.
The patent No. is the Chinese patent of ZL201210410047.6, disclose a kind of inducing mesenchymal stem cell to the method for Chondrocyte Differentiation and the application in osteoarthritis thereof, this invention uses little ball inducing culture Derived from Mesenchymal Stem Cells for without bracket cartilage, during use, the digestion of this cartilage is prepared into chondrocyte suspension and carries out intra-articular injection.Still there is the problem using single differentiation-inducing rear mescenchymal stem cell in this patent, Induction Process required time is long in addition, formation without bracket cartilage close structure, easily there is apoptosis in inner cell.
In sum, be not difficult to find out that the cell preparation cell type being applied to arthritis treatment is at present single, can not give full play to the biology performance of cell, method of inducing differentiation efficiency is lower.
Summary of the invention
The object of the invention is to overcome existing arthritis treatment preparation or method and have that cell preparation cell type is single, treatment time long, can not give full play to the biology performance of cell, differentiation-inducing efficiency is lower etc. is unfavorable for the problem of arthritic recovery.
For this reason, the invention provides a kind of cell preparation for the treatment of osteoarthritis, this cell preparation is applicable to the alleviation of inflammation and the reparation of damaged tissues in osteoarthritis disorders, realizes the alleviation of patients symptomatic and the recovery of function.
The cell preparation of above-mentioned treatment osteoarthritis, is made up of mescenchymal stem cell, the mescenchymal stem cell broken up to cartilage direction induction, Platelet-rich plasm and disperse medium.
Above-mentioned source for mesenchymal stem cells is any one in mesenchymal stem cells MSCs, fat mesenchymal stem cell, peripheral blood mescenchymal stem cell, Synovial Mesenchymal Stem Cells, umbilical cord mesenchymal stem cells and mesenchymal stem cells in umbilical cord blood.
The whole cell concentration of above-mentioned cell preparation is 10
5~ 10
8/ ml, wherein, described mescenchymal stem cell and described differentiation-inducing after mescenchymal stem cell ratio be 0.25:1 ~ 4:1.
In above-mentioned Platelet-rich plasm, PC is 1 ~ 2 × 10
6between/μ l.
The volume ratio of above-mentioned Platelet-rich plasm and disperse medium is at 0.5:1 ~ 2:1.
Above-mentioned disperse medium is any one in MEM, DMEM, D/F12, α-MDM, IMDM, BME, RPMI 1640 and McCoy's 5A culture medium.
Meanwhile, the invention provides a kind of method preparing the cell preparation of above-mentioned treatment osteoarthritis, comprise the following steps:
The separation and Culture of step one, mescenchymal stem cell: after isolate mesenchymal stem cells, is inoculated in cell culture medium, in 37 DEG C, volume fraction is the CO of 0.05%
2cultivate in incubator, change liquid every other day, until at the bottom of cell is paved with bottle 80% time, go down to posterity with 0.25% trypsinization;
The seed cell selected comprises the mescenchymal stem cell in various source, and described mescenchymal stem cell is any one in mesenchymal stem cells MSCs, fat mesenchymal stem cell, peripheral blood mescenchymal stem cell, Synovial Mesenchymal Stem Cells, umbilical cord mesenchymal stem cells, mesenchymal stem cells in umbilical cord blood;
Step 2: mescenchymal stem cell differentiation-inducing:
Get the above-mentioned mescenchymal stem cell being passaged to for 2 ~ 6 generations, be incubated in inductive differentiation medium, change liquid every other day, simultaneously, carry out 10 ~ 40kpa, 1Hz to mescenchymal stem cell, the compressive stress of 2 ~ 6h/d stimulates, by cellular morphology change, expression of enzymes, histochemical stain, histogenic immunity dyeing, molecular biology inspection, biochemical content analysis, to determine the differentiation of described mescenchymal stem cell;
Described inductive differentiation medium is DMEM in high glucose culture medium, wherein containing hyclone (FBS) 1 ~ 20%, transforming growth factor (TGF-β) 1 ~ 40ng/mL, vitamin C (Vc) 15 ~ 100 μ g/mL, Insulin 3 ~ 20 μ g/ml, transferrins 2 ~ 30 μ g/ml, Sodium Pyruvate 1 ~ 10mmol/L, dexamethasone 10
-7~ 10
-5mol/L, linoleic acid 2 ~ 30 μ g/mg, bovine serum albumin 0.5 ~ 10ng/ml;
Stimulate combined effect to carry out the differentiation-inducing of mescenchymal stem cell by inductive differentiation medium and compressive stress, with the secretion of the expression and proteoglycan that promote cartilage related gene, and then be beneficial to the differentiation of described mescenchymal stem cell to cartilage direction;
Step 3: the preparation of Platelet-rich plasm: get the venous blood adding anticoagulant, centrifuging Platelet Concentrate, obtaining PC is 1 ~ 2 × 10
6the blood plasma of/μ l;
Step 4: the preparation of cell preparation
Mescenchymal stem cell and differentiation-inducing mescenchymal stem cell are digested and count stand-by, mixed with the volume ratio of 0.5:1 ~ 2:1 with disperse medium by Platelet-rich plasm, afterwards with this stand-by cell that suspends, adjusting whole cell concentration is 10
5~ 10
8/ ml, wherein mescenchymal stem cell and differentiation-inducing after mescenchymal stem cell ratio be 0.25:1 ~ 4:1;
Above-mentioned disperse medium is any one in MEM, DMEM, D/F12, α-MDM, IMDM, BME, RPMI 1640 and McCoy's 5A culture medium;
Culture medium can provide basal nutrient for the existence of cell and breeding, and containing abundant somatomedin in Platelet-rich plasm, these somatomedin are in cell proliferation and differentiation, play a significant role in tissue repair regeneration, by cell, Platelet-rich plasm and culture medium three compound, respective performance be can effectively play, arthritic remission and tissue repair realized.
Compared with existing product and technology, the present invention has the following advantages:
(1) by mescenchymal stem cell and differentiation-inducing after mescenchymal stem cell used in combination, both can play the immunoloregulation function of mescenchymal stem cell, improve articular cavity microenvironment, be beneficial to the alleviation of inflammation and the reparation of damage; The mescenchymal stem cell broken up can be made again after implanting namely to start process of tissue reparation, shorten reparation process.Two kinds of mixing with cells use and can at utmost play respective biological availability, promote reparation and the regeneration of tissue.In addition be compounded with Platelet-rich plasm composition, its multiple somatomedin and active component are beneficial to reparation and the agglutination of damaged tissue.
(2) inductive differentiation medium and compressive stress is adopted to stimulate two kinds of methods combining to carry out the differentiation of mesenchymal stem cells into chondrocytes, the expression of cartilage related gene and the secretion of proteoglycan can be promoted, improve induced efficiency, shorten differentiation-inducing required time, simply efficiently.Cellular morphology observation, Toluidine blue staining and GAG content detection display induction can generate hyaline cartilage cell after 7 days.
(3) cell preparation prepared by is suspensions, can ensure that cell and various somatomedin, active substance disperse in whole articular cavity, can improve articular cavity microenvironment, be beneficial to arthritic recovery after intraarticular injection.Results of animal shows cell preparation prepared by the present invention substantially can realize arthritic reparation 8 weeks time, neocartilage surface is comparatively smooth, organizational structure is comparatively obviously clear, and the coarse injustice of blank group cartilage surface do not processed, destructurized.
accompanying drawing illustrates:
Figure 1A is differentiation-inducing mescenchymal stem cell cellular morphology figure.
Figure 1B is without differentiation-inducing mescenchymal stem cell cellular morphology figure.
Fig. 1 C is using mescenchymal stem cell as negative control, using chondrocyte as positive control, gets its supernatant respectively and carries out GAG content detection result figure.
Fig. 2 A be experimental group within 8 weeks, draw materials Histological section figure.
Fig. 2 B is normal cartilage slice map.
Fig. 2 C is the Histological section figure of blank group.
Detailed description of the invention
Have that cell preparation cell type is single, treatment time long, can not give full play to the biology performance of cell to overcome existing arthritis treatment preparation or method, differentiation-inducing efficiency is lower etc. is unfavorable for the problem of arthritic recovery, present embodiments provide a kind of cell preparation for the treatment of osteoarthritis, this cell preparation is applicable to the alleviation of inflammation and the reparation of damaged tissues in osteoarthritis disorders, realizes the alleviation of patients symptomatic and the recovery of function.
The cell preparation for the treatment of osteoarthritis, comprises cell, Platelet-rich plasm and disperse medium.The cell wherein related to comprises mescenchymal stem cell and the mescenchymal stem cell two kinds of cell types to the differentiation of cartilage direction induction.
Source for mesenchymal stem cells comprises mesenchymal stem cells MSCs, fat mesenchymal stem cell, peripheral blood mescenchymal stem cell, Synovial Mesenchymal Stem Cells, umbilical cord mesenchymal stem cells, mesenchymal stem cells in umbilical cord blood etc.
In the Platelet-rich plasm used, PC is 1 ~ 2 × 10
6between/μ l.The volume ratio of Platelet-rich plasm and disperse medium is between 0.5:1 ~ 2:1.
Disperse medium refers to conventional cell culture medium, include but not limited to MEM, DMEM, D/F12, α-MDM, IMDM, BME, RPMI 1640, McCoy's 5A culture medium, for the active component in suspension cell and Platelet-rich plasm and various cell growth factor, and provide nutrition.
The preparation of the cell preparation of above-mentioned treatment osteoarthritis comprises: the separation and Culture of mescenchymal stem cell; Mescenchymal stem cell differentiation-inducing; The preparation of Platelet-rich plasm; The preparation of cell preparation, so far then complete the preparation of the cell preparation to treatment osteoarthritis, concrete preparation process is:
Step one: the separation and Culture of mescenchymal stem cell:
The seed cell selected comprises the mescenchymal stem cell in various source, this mescenchymal stem cell can mesenchymal stem cells MSCs, fat mesenchymal stem cell, peripheral blood mescenchymal stem cell, Synovial Mesenchymal Stem Cells, umbilical cord mesenchymal stem cells, any one in mesenchymal stem cells in umbilical cord blood, the cultural method of seed cell and the cell culture medium of use can with reference to pertinent literature (Hu Bin, Liu Xiaohua, Zhao Lin. the Isolation and identification of mesenchymal stem cells MSCs. Chinese Tissue Engineering Study and clinical rehabilitation, 2011,15(27): 5108 ~ 5111; Wang Juan, Lu Yan, He Dongmei. human umbilical cord mesenchymal stem cells in-vitro separation, Isolation and characterization. Ji'nan University's journal, 2009,30(4): 367 ~ 372; Zhang Yueshuan, Wei Hua, Ma Jian. the improvement of Umbilical cord blood mesenchymal stem cells isolated culture method. Tongji University's journal (medicine), 2010,31(1): 27 ~ 31; Wang Dongmei, Shi Bei, Zhao Ranzun, etc. the separation and Culture of. rabbit peripheral blood mescenchymal stem cell and frozen. Zunyi Medical College's journal, 2007,30(3): 336 ~ 338. positive political affairs, Li Weiping, Yang Rui, etc. Synovial Mesenchymal Stem Cells separation and Culture, immunophenotypic characterization and the depression effect in mixing lymph reaction system. Chinese Tissue Engineering Study, 2012,16(19): 3515 ~ 3519).Specifically: after isolate mesenchymal stem cells, be inoculated in cell culture medium, in 37 DEG C, volume fraction is the CO of 0.05%
2cultivate in incubator, change liquid every other day, until at the bottom of cell is paved with bottle 80% time, go down to posterity with 0.25% trypsinization.
Step 2: mescenchymal stem cell differentiation-inducing:
Get the above-mentioned mescenchymal stem cell being passaged to for 2 ~ 6 generations, be incubated in inductive differentiation medium, change liquid every other day.Meanwhile, carry out 10 ~ 40kpa, 1Hz to cell, the compressive stress of 2 ~ 6h/d stimulates.Changed by cellular morphology, expression of enzymes, histochemical stain, histogenic immunity dyes, and molecular biology is checked, the differentiation of the method determination cells such as biochemical content analysis.
Above-mentioned inductive differentiation medium is DMEM in high glucose culture medium, wherein containing hyclone (FBS) 1 ~ 20%, transforming growth factor (TGF-β) 1 ~ 40ng/mL, vitamin C (Vc) 15 ~ 100 μ g/mL, Insulin 3 ~ 20 μ g/ml, transferrins 2 ~ 30 μ g/ml, Sodium Pyruvate 1 ~ 10mmol/L, dexamethasone 10
-7~ 10
-5mol/L, linoleic acid 2 ~ 30 μ g/mg, bovine serum albumin 0.5 ~ 10ng/ml;
This step adopts inductive differentiation medium and compressive stress to stimulate combined effect to carry out the differentiation-inducing of mescenchymal stem cell, can promote the expression of cartilage related gene and the secretion of proteoglycan, be beneficial to the differentiation of mescenchymal stem cell to cartilage direction.
Step 3: the preparation of Platelet-rich plasm:
The preparation of Platelet-rich plasm can list of references method carry out (Liu Jinghan, Ou Yangxilin, Shi Qun, etc. in Platelet-rich plasm preparation process, platelet CD62p expresses. Chinese experimental hematology magazine, 2002,10(3): 253 ~ 256; Jin Zhe, Lv Gang, Wang Yuxiang. secondary centrifuging legal system is for the comparison of Platelet-rich plasm centrifugal condition. Chinese Medical Sciences University's journal, 2012,41(3): 237 ~ 240).Can be specifically: get the venous blood adding anticoagulant, centrifuging Platelet Concentrate that obtaining PC is 1 ~ 2 × 10
6the blood plasma of/μ l.
Step 4: the preparation of cell preparation
Mescenchymal stem cell and differentiation-inducing mescenchymal stem cell are digested and count stand-by, mixed with the ratio of 0.5:1 ~ 2:1 with disperse medium by Platelet-rich plasm, afterwards with this stand-by cell that suspends, adjusting whole cell concentration is 10
5~ 10
8/ ml, wherein mescenchymal stem cell and differentiation-inducing after mescenchymal stem cell ratio be 0.25:1 ~ 4:1.Disperse medium used includes but not limited to MEM, DMEM, D/F12, α-MDM, IMDM, BME, RPMI 1640, McCoy's 5A culture medium.
Below by way of some examples, the cell preparation of above-mentioned treatment osteoarthritis and preparation thereof are described in further detail.
Embodiment 1: fat mesenchymal stem cell is seed cell
The separation and Culture of step one, fat mesenchymal stem cell
By new zealand rabbit general anaesthesia, obtain fatty tissue from subcutaneous abdomen, rinse 3 times with PBS solution, remove blood vessel and connective tissue, shred, add the type i collagen enzyme of 3 times of volumes, 37 DEG C of digestion 2 h, do not digest bulk fat tissue completely with 200 mesh filter screen eliminations, centrifugally abandon supernatant, with PBS re-suspended cell, clean 3 times, add the resuspended sedimentation cell of low sugar DMEM containing 10%FBS afterwards, be inoculated in plate, in 37 DEG C, volume fraction is the CO of 0.05%
2cultivate in incubator, change liquid every other day, until at the bottom of cell is paved with bottle 80% time, go down to posterity with 0.25% trypsinization.
Step 2, fat mesenchymal stem cell differentiation-inducing
Get P5 fat subsitutes mescenchymal stem cell with 10
6/ ml is seeded in culture dish, and inductive differentiation medium is DMEM in high glucose culture medium, containing 15% hyclone, 10 μ g/L TGF-β 1,20mg/L Vc, 5mg/L insulin, 6.25mg/L transferrins, 5mmol/L Sodium Pyruvate, 10
-7mol/L dexamethasone, 5 μ g/mg linoleic acids, 2ng/ml bovine serum albumin, change liquid every other day, and applying compressive stress with 40Kpa, 1Hz, 2h/d to cultured cell stimulates, and what complete fat mesenchymal stem cell after 7 days is differentiation-inducing.
The preparation of step 3, Platelet-rich plasm
Get peripheral blood 5ml, add anticoagulant, centrifugal 30 minutes with 900 revs/min, be separated blood plasma near platelet and leukocytic cream gently and obtain Platelet-rich plasm.Gained PC is 1.14 × 10
6/ μ l.
The preparation of step 4, cell preparation
Mescenchymal stem cell and differentiation-inducing mescenchymal stem cell are digested and count stand-by, mixed with the volume ratio of 1:2 with MEM culture medium by Platelet-rich plasm, afterwards with this stand-by cell that suspends, adjusting whole cell concentration is 10
8/ ml, wherein mescenchymal stem cell and differentiation-inducing after mescenchymal stem cell ratio be 1:4.So far the preparation of the cell preparation of completed treatment osteoarthritis.
Fig. 1 shows the fat mesenchymal stem cell testing result after differentiation-inducing 7 days, cellular morphology observed result shows, by having presented polygonal chondrocyte feature (see Figure 1A) to cell after differentiation-inducing 7 days of fat mesenchymal stem cell, and the fat mesenchymal stem cell not doing differentiation-inducing process is still in fibrous Spiral distribution (see Figure 1B); Get its supernatant simultaneously and carry out GAG detection, using mescenchymal stem cell as negative control, using chondrocyte as positive control, the mescenchymal stem cell that result also shows differentiation-inducing process can secrete more glycosaminoglycans, demonstrate chondrocyte characteristic (see Fig. 1 C), illustrate that fat mesenchymal stem cell is successfully induced as chondrocyte.
Fig. 2 display be the Histological section that the cell preparation of the treatment osteoarthritis prepared by above-mentioned steps is repaired arthritis and drawn materials for 8 weeks, result as shown in Figure 2 A, display test group can realize arthritic reparation, reparation smooth surface is smooth, and cartilage structure is close to the normal cartilage shown in Fig. 2 B, and blank group as shown in Figure 2 C, cartilage surface is coarse, tide line is not obvious, cartilage structure is damaged.
Embodiment 2: human umbilical cord mesenchymal stem cells is seed cell is that umbilical cord derives from donor and contributes.
The separation and Culture of step one, umbilical cord mesenchymal stem cells:
Umbilical cord tissue is taken out from collecting bottle, is placed in ethanol cleaning and proceeds to aseptic PBS removal connective tissue, congestion etc. for 2 times, fully clean afterwards, remove blood stains.Being proceeded to by umbilical cord in large size culture dish, be longitudinally separated after cutting off into 3-4cm length, is that umbilical cord tissue tubulose becomes lamellar.Lamellar umbilical cord tissue is further separated, is cut into 2-3mm
3the piece of tissue of size, piece of tissue planted one by one in advance with in the culture dish of the D/F12 culture medium bag quilt containing 20% hyclone, be placed in 37 DEG C, volume fraction is 5%CO
2in saturated humidity incubator, every 24 lab scales add 0.5ml culture medium, and after 72 hours, full dose changes liquid, change weekly liquid 2 times, Growth of Cells merge only about 80% time go down to posterity.
Step 2: umbilical cord mesenchymal stem cells differentiation-inducing
Get P2 for umbilical cord mesenchymal stem cells with 10
5/ ml is seeded in culture dish, and inductive differentiation medium is DMEM in high glucose culture medium, wherein containing 1% hyclone, 30 μ g/L TGF-β 3,80mg/L Vc, 15mg/L insulin, 20mg/L transferrins, 2mmol/L Sodium Pyruvate, 10
-5mol/L dexamethasone, 25 μ g/mg linoleic acids, 6ng/ml bovine serum albumin, change liquid every other day.Apply compressive stress with 10Kpa, 1Hz, 6h/d to cultured cell to stimulate.Complete differentiation-inducing after 7 days.
The preparation of step 3, Platelet-rich plasm
Get peripheral blood 5ml, add anticoagulant, centrifugal 10 minutes with 1400 revs/min for the first time, carefully draw top blood plasma afterwards and carry out secondary centrifuging, secondary centrifuging parameter be 1400 revs/min centrifugal 15 minutes, remove supernatant gently, obtain bottom centrifugal liquid be Platelet-rich plasm.Gained PC is 1.96 × 10
6/ μ l.
The preparation of step 4, cell preparation
Mescenchymal stem cell and differentiation-inducing mescenchymal stem cell are digested and count stand-by, mixed with the volume ratio of 2:1 with D/F12 culture medium by Platelet-rich plasm, afterwards with this stand-by cell that suspends, adjusting whole cell concentration is 10
6/ ml, wherein mescenchymal stem cell and differentiation-inducing after mescenchymal stem cell ratio be 3:1.So far the preparation of the cell preparation of completed treatment osteoarthritis.
Claims (9)
1. treat a cell preparation for osteoarthritis, it is characterized in that: by mescenchymal stem cell, formed by mescenchymal stem cell, Platelet-rich plasm and the disperse medium broken up to cartilage direction induction under inductive differentiation medium and compressive stress stimulation combined effect.
2. cell preparation according to claim 1, is characterized in that: described source for mesenchymal stem cells is any one in mesenchymal stem cells MSCs, fat mesenchymal stem cell, peripheral blood mescenchymal stem cell, Synovial Mesenchymal Stem Cells, umbilical cord mesenchymal stem cells and mesenchymal stem cells in umbilical cord blood.
3. cell preparation according to claim 1, is characterized in that: the whole cell concentration of described cell preparation is 10
5~ 10
8/ ml, wherein, described mescenchymal stem cell and described differentiation-inducing after mescenchymal stem cell ratio be 0.25:1 ~ 4:1.
4. cell preparation according to claim 1, is characterized in that: in described Platelet-rich plasm, PC is 1 ~ 2 × 10
6/ μ l.
5. cell preparation according to claim 1, is characterized in that: the volume ratio of described Platelet-rich plasm and disperse medium is 0.5:1 ~ 2:1.
6. cell preparation according to claim 1, is characterized in that: described disperse medium is any one in MEM, DMEM, D/F12, α-MDM, IMDM, BME, RPMI 1640 and McCoy's 5A culture medium.
7. a preparation method for the cell preparation for the treatment of osteoarthritis according to claim 1, is characterized in that, comprise the following steps:
The separation and Culture of step one, mescenchymal stem cell;
After isolate mesenchymal stem cells, be inoculated in cell culture medium, in 37 DEG C, volume fraction is the CO of 0.05%
2cultivate in incubator, change liquid every other day, until at the bottom of cell is paved with bottle 80% time, go down to posterity with 0.25% trypsinization;
Described mescenchymal stem cell is any one in bone marrow mescenchymal stem cell, adipose tissue-derived mesenchymal stem cells, peripheral blood source mescenchymal stem cell, synovial membrane source mescenchymal stem cell, Mesenchymal Stem Cells from Umbilical Cord and Cord blood source mescenchymal stem cell;
Step 2: mescenchymal stem cell differentiation-inducing
Get the above-mentioned mescenchymal stem cell being passaged to for 2 ~ 6 generations, be incubated in inductive differentiation medium, change liquid every other day, simultaneously, carry out 10 ~ 40kpa, 1Hz to mescenchymal stem cell, the compressive stress of 2 ~ 6h/d stimulates, by cellular morphology change, expression of enzymes, histochemical stain, histogenic immunity dyeing, molecular biology inspection, biochemical content analysis, to determine the differentiation of described mescenchymal stem cell;
Described inductive differentiation medium is DMEM in high glucose culture medium, wherein containing hyclone FBS 1 ~ 20%, transforming growth factor β_1 ~ 40ng/mL, vitamin C 15 ~ 100 μ g/mL, Insulin 3 ~ 20 μ g/ml, transferrins 2 ~ 30 μ g/ml, Sodium Pyruvate 1 ~ 10mmol/L, dexamethasone 10
-7~ 10
-5mol/L, linoleic acid 2 ~ 30 μ g/mg, bovine serum albumin 0.5 ~ 10ng/ml;
Stimulate combined effect to carry out the differentiation-inducing of mescenchymal stem cell by inductive differentiation medium and compressive stress, with the secretion of the expression and proteoglycan that promote cartilage related gene, and then be beneficial to the differentiation of described mescenchymal stem cell to cartilage direction;
Step 3: hematoblastic blood plasma is rich in preparation, gets the venous blood adding anticoagulant, centrifuging Platelet Concentrate, obtaining PC is 1 ~ 2 × 10
6the blood plasma of/μ l;
Step 4: the preparation of cell preparation
Mescenchymal stem cell and differentiation-inducing mescenchymal stem cell are digested and count stand-by, mixed with the volume ratio of 0.5:1 ~ 2:1 with disperse medium by Platelet-rich plasm, afterwards with this stand-by cell that suspends, adjusting whole cell concentration is 10
5~ 10
8/ ml, wherein mescenchymal stem cell and differentiation-inducing after mescenchymal stem cell ratio be 0.25:1 ~ 4:1.
8. the preparation method of the cell preparation for the treatment of osteoarthritis as claimed in claim 1, it is characterized in that, the volume ratio of described Platelet-rich plasm and disperse medium is 0.5:1 ~ 2:1.
9. the preparation method of the cell preparation for the treatment of osteoarthritis as claimed in claim 1, is characterized in that, disperse medium used is any one in MEM, DMEM, D/F12, α-MDM, IMDM, BME, RPMI 1640 and McCoy's 5A culture medium.
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