CN104771413A - Application of arthroscopic flushing fluid sourced mesenchymal stem cells in regenerative repair - Google Patents
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Abstract
The invention discloses application of arthroscopic flushing fluid sourced mesenchymal stem cells in regenerative repair, a large number of mesenchymal stem cell liquids which are obtained by an arthroscopic surgery and sourced from articular tissues such as cartilaginous tissue, synovium and synovial fluid), cell components in the mesenchymal stem cell liquids are enriched by piecewise collection and membrane filtration and other processes for cultivation and amplification; after wall adherent growth, proliferation, differentiation and exogenous microbial contamination detection and other procedures of the enriched cells, according to the international ISCT (International Society for Cellular Therapy) recommended mesenchymal stem cell criteria, purity and homogeneity of the enriched stem cells are identified and analyzed, and finally the enriched cells are respectively stored in a master cell bank and a working cell bank for cell therapy. According to the application, arthroscopic surgery discarded liquor can be fully used for separation of the stem cells for treatment of patients.
Description
Art
Applied the present invention relates to a kind of arthroscope flushing liquor derived mesenchymal stem cell in Regeneration and Repair, and the application on the separation method and Regeneration and Repair treatment there is provided a kind of arthroscope flushing liquor derived mesenchymal stem cell, belong to medicine technology field.
Background technology
Arthroscope is a kind of diameter 5mm for observing intra articular structure or so bar-shaped optical instrument, is the endoscope for diagnosis and treatment joint diseases.Apparatus popularization and application since 1970.Tubule, equipped with a lens, is inserted intra articular, the structure of intra articular will be shown on a monitor by arthroscope in the end of a tubule.It therefore, it can observe directly the structure of intra articular.Arthroscope is applied not only to the diagnosis of disease, and is widely used for the treatment of joint disease.Arthrocsopic surgery is a kind of Minimally Invasive Surgery, starts to be mainly used in knee joint, and rear sequential use is in hip joint, shoulder joint, ankle-joint, Minor articulus such as elbow joint and finger etc..
As endoscopic technic is gradually popularized in China, arthrocsopic surgery has turned into the conventional diagnosis and treatment means of Bones and joints surgery.So far in each system scope of whole body, arthroscope function is most comprehensive, most widely used, and is consequently formed independent arthroscopic surgery.Arthrocsopic surgery business turns into the growth point of hospital and section office's performance.
2008, China had 790 hospitals and possesses arthroscope system facility, it is possible to implement arthrocsopic surgery, these hospitals are concentrated mainly on Grade A hospital；By 2012, China had 2640 hospitals and possesses arthroscope system facility, it is possible to implement arthrocsopic surgery, average annual growth rate 35%, covered almost all of tertiary hospitals and about 30% diformazan hospital.The budget of hospital's buying arthroscope system is simultaneously not affected by the influence that economic crisis and China's economic are slowed down.In some flourishing cities such as Beijing, Shanghai, its arthroscopic techniques level has been able to match in excellence or beauty with developed country.Arthrocsopic surgery has begun to turn to basic unit from city tertiary hospitals, is adapted to China's national situation, crowd is covered to greatest extent.
Arthrocsopic surgery can treat intra-articular various inflammation such as Osteoarthritis synovitis, traumatic arthritis, rheumatoid arthritis, tuberculous arthritis, pyogenic arthritis, osteochondritis dissecans etc., and synovial chondromatosis；Chondromalacia of patella；Spur (spur), episome, synovial membrane wrinkle wall, joint disorders disease, meniscus injury, intracapsular adhesion, various intra-articular fractures, each portion's synarthrophysis and joint motion are limited, the arthralgia of various unknown causes.
Arthrocsopic surgery is compared with arthrotomy, with following advantages：Otch is small, attractive in appearance, can avoid late period irritation because of caused by the scar of articular surface and motive position；Belong to Minimally Invasive Surgery, pain is small, and after-operation response is smaller, patient is easy to receive；Early postoperation is movable and uses limbs, it is to avoid long-term bed complication, reduces nursing staff and expense；Complication is relatively fewer；Periarticular muscles structure is had substantially no effect on, it is postoperative to carry out functional training in early days, prevent joint fixed for a long time caused useless use and complication；O＆E can be carried out to intra-articular lesion under intimate physiological environment, there is the title of " eyes and finger being put into intra-articular ", dynamic property inspection can be carried out to joint, improve diagnosis capability, some diseases such as plica syndrome, is just established by arthroscope；Arthroscope can implement the operation that conventional open operation is difficult to complete, such as partial meniscectomy art.Contraindication is few, such as physical qualification difference can not row routine operation, but not necessarily avoid arthrocsopic surgery.
Arthroscope also has some shortcomings, and one is that not each doctor patiently carries out arthrocsopic surgery, because need to be operated through small otch with the apparatus of fine and rapid wear.Two be that operation apparatus may produce noticeable wear and cut to articular cartilage in nervous joint space, particular without the doctor of experience.Three be possible time consuming in initially use arthroscopic procedures.Four be that special equipment is numerous and diverse and expensive.
Publication No. CN103796659A Chinese patent discloses a kind of cartilage cell's therapeutant containing collagen, derivatives of hyaluronic acids and mammal umbilical cord Derived Stem Cells, is related to a kind of medical science plyability biomaterial.More particularly it relates to a kind of medical science plyability biomaterial comprising collagen and derivatives of hyaluronic acids.Further, the present invention relates to a kind of chondrocyte therapeutic, it uses the biomaterial and the stem cell from mammal umbilical cord.The biomaterial does not cause immune response, with superior persistence, and the chondrocyte therapeutic comprising the biomaterial and stem cell makes it possible to carry out arthrocsopic surgery, thus reduce the pain of patient and effectively treat degenerative arthritis and cartilage damage.
Publication No. CN103031271A Chinese patent discloses a kind of quick method for isolating and purifying embryo's stem cell of cranial nerve, 3 D stereo culture is carried out to nerve cell using hot-cast socket polymer NIPA and polyethylene glycol, the characteristic that can breed using NSC in gel environment and form neural ball reaches the purpose isolated and purified to NSC.The neural ball warp immunofluorescence label of formation is determined, and is shown that about 93% cell is positive for Nestin, is illustrated that the method separation and purification of nerve stem cell is easy and effective.The embryo neural stem cells isolated and purified through the method can be differentiated to form neuron, oligodendroglia and Deiter's cells in vitro.
Publication No. CN102676451A Chinese patent discloses a kind of method of the separating mesenchymal stem cell from placenta, and this method comprises the following steps：(a) placental lobules is taken, is fully rinsed with PBS, to remove the blood remained in placenta；(b) placental lobules is cut into bulk, adds the PBS containing tissue digestion enzyme, then digestion is incubated at 37 DEG C；(c) tissue block is filtered with copper mesh, grinds to promote filtering if necessary；(d) filtered fluid of collection is centrifuged, separates mononuclearcell, then with the obtained cell of MSC culture mediums suspension, then in 37 DEG C, 5%CO2Cultivated in incubator；(e) after after disseminated cell formation clone, each clone cell of picking is cultivated, after after cell fusion, using pancreatin had digestive transfer culture, produces placenta mesenchyma stem cell respectively with MSC culture mediums；And optional following one or more steps：At least one of (f) placenta mesenchyma stem cell obtained by step (e), detection following items：Cytoactive, cell contamination, hereditary disease, HLA-ABC/DR distribution type；(g) placenta mesenchyma stem cell after being passed on obtained by step (e) is frozen in liquid nitrogen；(h) database of the placenta stem-cell comprising information above is set up, and is associated the database and the freeze-stored cell of step (g).
Document above data shows, each to joint tissue such as cartilaginous tissue, synovial membrane, synovia, produced a large amount of flushing liquors do not make full use of when carrying out arthrocsopic surgery, directly abandoned in surgical procedure, and the stem cell needed for performing the operation is achieved other ways, although the stem cell rejection obtained is low, surgery cost can be caused higher.
The content of the invention
The technical solution adopted by the present invention includes：Arthrocsopic surgery of learning from else's experience obtains the mescenchymal stem cell liquid that (cartilaginous tissue, synovial membrane, synovia) is largely respectively organized from joint, using programs such as Fractional Collections, membrane filtrations, and enrichment wherein cell component is cultivated and expanded；The cell of enrichment is after the programs such as adherent growth, propagation, differentiation and inoculating microbe pollution detection, the mescenchymal stem cell standard that reference world ISCT recommends is to the stem cell purity and the identification and analysis of homogeneity of enrichment, and being finally separately stored in master cell bank and working cardial cell storehouse is used for cell therapy.
Therefore, the present invention provides arthroscope flushing liquor derived mesenchymal stem cell and applied in Regeneration and Repair, and step includes：
(1) joint fluid is collected：Fractional Collections flushing liquor is carried out to flushing liquor in arthroscopic surgical procedures with sterile collecting pipe, saved backup；
(2) it is prepared by stem cell separation：By Fractional Collections flushing liquor by ceramic membrane filter, cell enrichment is carried out, gained cell is separately added into after nutrient solution and suspended again, by 3 × 105/ ml concentration is inoculated into blake bottle and is placed with Secondary Culture in 12 orifice plates of sterilization cover glass in advance, and nutrient solution is the L-DMEM nutrient solutions containing 15%FBS, and 5~7d of culture is removed after not adherent cell, obtains attached cell；
(3) qualified donor material archives：The identification and analysis of purity and homogeneity is carried out to attached cell with reference to international mescenchymal stem cell standard, qualified mescenchymal stem cell is determined, sets up associated profiles, while being passed on to qualified stem cell, 4~5d is passed on once；
(4) mescenchymal stem cell into cartilage differentiation：By the subculture stem cell of more than 3 times, it is 4 × 10 that concentration, which is made,5/ ml suspension, 1ml cell suspensions are taken in 15m1 plastic centrifuge tubes, 500g centrifuges 15min, forms it into cell micelle, carefully sucks nutrient solution, add 2ml serum-free chondrocyte induction nutrient solutions, it is subsequently placed in CO2gas incubator and cultivates, changes liquid first after 4 days, changed liquid every 2 days later, the cell mass of culture is taken out after cultivating 21 days, cartilage differentiation effect is detected into；
(5) it is implanted into Regeneration and Repair effect：Qualified mescenchymal stem cell is implanted at patient articular by finite concentration, regeneration of joints repairing effect is observed.
In a specific embodiment, step (1) described preservation condition is that aseptically, 4 DEG C of storages are less than 4 hours, are maintained in transportation under 4 DEG C of environment.
In a specific embodiment, cell culture environment condition described in step poly- (2) and (4) is 37 DEG C, 5%CO2, saturated humidity CO2gas incubator in cultivate.
In a specific embodiment, walk serum-free chondrocyte induction nutrient solution described in poly- (4) and refer to DMEM in high glucose (H-DMEM) 11ng/mL containing TGF-β, dexamethasone 10- 7Mmol/L, vitamin C 50mg/L, insulin 6.25ng/L, transferrins 6.25ug/mL, bovine serum albumin(BSA) 1.25ug/mL.
Described international mesenchymal stem cells standard refers to mescenchymal stem cell standard such as CD44, CD90, CD105, CD147, CD271, CD166 recommended with reference to international cell therapy association (ISCT) etc..
In a specific embodiment, implantation concentration of stem cells described in poly- (5) is walked 50~4 × 107/ml。
Technique effect
1st, the present invention makes full use of the mescenchymal stem cell in the arthroscope flushing liquor of discarding, for the Regeneration and Repair treatment for articular cartilage lesion.
2nd, a large amount of stem cells can be obtained by being recycled by discarded object, the need for this will meet patient significantly to stem cell, decrease the probability for employing patient other parts stem cell, from second operation injury.
3rd, the medical treatment cost of patient can be greatly reduced by the mescenchymal stem cell in arthroscope flushing liquor by large-scale culture, improves the success rate of operation.
4th, by the way of directly culture arthrocsopic surgery flushing liquor, red blood cell is opened without by centrifuging, the influence of mescenchymal stem cell can be greatly reduced.
Brief description of the drawings
Fig. 1, the mescenchymal stem cell P0 of culture are for the aspect graph under light microscopic；
Fig. 2, the mescenchymal stem cell P1 of culture are for the aspect graph under light microscopic；
Fig. 3, the mescenchymal stem cell P2 of culture are for the aspect graph under light microscopic；
Fig. 4, the mescenchymal stem cell P3 of culture are for the aspect graph under light microscopic；
Fig. 5, the alcian blue of mescenchymal stem cell into the cartilage differentiation of culture are dyed.
Embodiment
Below, the present invention will be further detailed with embodiment, but it is not limited to any one or similar example of these embodiments.
Embodiment 1：
Fractional Collections flushing liquor is carried out to flushing liquor in arthroscopic surgical procedures with sterile collecting pipe, aseptically, 4 DEG C of storages are less than 4 hours, are maintained in transportation under 4 DEG C of environment.By Fractional Collections flushing liquor by 0.22 μm of ceramic membrane filter, cell enrichment is carried out, is suspended again after adding nutrient solution, by 3 × 105/ ml concentration is inoculated into blake bottle and is placed with advance in 12 orifice plates of sterilization cover glass and cultivates, 37 DEG C, 5%CO2, saturated humidity CO2gas incubator in cultivate, nutrient solution is the L-DMEM nutrient solutions containing 15%FBS, and 5~7d of culture is removed after not adherent cell, obtains attached cell.
Mescenchymal stem cell standard such as CD44, CD90, CD105, CD147, CD271, CD166 recommended with reference to international cell therapy association (ISCT) etc., the identification and analysis of purity and homogeneity is carried out to attached cell, determine qualified mescenchymal stem cell, set up associated profiles, qualified stem cell is passed on simultaneously, Fig. 1,2,3,4 are once shown in 4~5d passages.
By the subculture stem cell of more than 3 times, it is 4 × 10 that concentration, which is made,5/ ml suspension, 1ml cell suspensions are taken in 15m1 plastic centrifuge tubes, 500g centrifuges 15min, form it into cell micelle, carefully suck nutrient solution, 2ml serum-free chondrocyte induction nutrient solutions are added, serum-free chondrocyte induction nutrient solution refers to DMEM in high glucose (H-DMEM) 11ng/mL containing TGF-β, dexamethasone 10- 7Mmol/L, vitamin C 50mg/L, insulin 6.25ng/L, transferrins 6.25ug/mL, bovine serum albumin(BSA) 1.25ug/mL.It is subsequently placed in 37 DEG C, 5%CO2, saturated humidity CO2gas incubator in cultivate, change liquid first after 4 days, changed liquid every 2 days later, take out the cell mass of culture after 21 days in culture, detect into cartilage differentiation effect, see Fig. 5.
Qualified mescenchymal stem cell is pressed 50~4 × 107/ ml is implanted directly at patient articular, observes regeneration of joints repairing effect, wherein there is 9 patient's repairing effects very well, 1 patient's repairing effect is not obvious.
Into cartilage differentiation index：
1) proteoglycan Alcian Blue (pungent indigo plant difficult to understand) are dyed：The main content for checking acidic mucopolysaccharide (chondroitin sulfate) in cartilaginous tissue and chondrocyte matrix.Cytoplasm dyes blueness during Alcian blue stained positives, and nucleus is red, calculates positive cell, removes mean value calculation positive rate.Step is as follows：Mescenchymal stem cell cell mass of the Fiber differentiation after 21 days is taken, dewaxing is to water for paraffin section (making same 2.2.1), and distilled water embathes 3 times；5min is incubated with alcian blue, flowing water rinses 2min, and Maxwell haematoxylin redyes 5min, and flowing water rinses 2min, dried, the proteoglycans deposition of observation of cell under microscope.
2) the collagen immunization chemical stainings of Type- II：Positive performance is that brown yellow granule is distributed in cytoplasm.SABC methods:2 times each 5 minutes → use distilled water is washed in dewaxing, aquation → PBS or PBS prepares fresh 3%H2O2, room temperature is closed 5-10 minutes, 5 minutes → dropwise addition Normal Goat Serum confining liquid is washed with 3 times → antigen retrieval → PBS of distillation washing, room temperature 20 minutes, get rid of surplus liquid → dropwise addition Ι and resist 50 μ l, it is stored at room temperature 1 hour or 4 DEG C overnight or 37 DEG C of 1 hour → PBS washes 3 times each 2 minutes → dropwise addition biotinylation II and resisted, 20 DEG C of -37 DEG C of 20 minutes → PBS washes 3 times each 2 minutes → dropwise addition, 20 DEG C of -30 DEG C of 20 minutes → PBS of reagent SABC and wash 4 each 5 minutes → DAB colour developings：Kit or autogamy chromogenic reagent → dehydration, transparent, mounting, microscopy.
3) qRT-PCR detects that SOX9, Aggrecan, the mRNA of II Collagen Type VI determine (method is ibid)；Primer synthesis is as follows required for reaction：
4) electron microscopic observation：Mescenchymal stem cell of the Fiber differentiation after 14 days is taken, cell climbing sheet is taken out and is rinsed 2 times with PBS, to remove nutrient solution, 3-4% glutaraldehydes fix 3h, and 1% osmium tetroxide is fixed, conventional ethanol serial dehydration after rinsing, and point of proximity is dried, scanning electron microscopic observation after metal spraying.The cell in blake bottle is collected simultaneously, is collected in after digestion in 1.5ml centrifuge tube, and 3-4% glutaraldehydes fix 24h, ultra-thin section, transmission electron microscope observing cell ultrastructure and cellular matrix formational situation.
Claims (5)
1. the present invention provides arthroscope flushing liquor derived mesenchymal stem cell and applied in Regeneration and Repair, step includes：
(1) joint fluid is collected：Fractional Collections flushing liquor is carried out to flushing liquor in arthroscopic surgical procedures with sterile collecting pipe, saved backup；
(2) it is prepared by stem cell separation：By Fractional Collections flushing liquor by ceramic membrane filter, cell enrichment is carried out, gained cell is separately added into after nutrient solution and suspended again, by 3 × 105/ ml concentration is inoculated into blake bottle and is placed with Secondary Culture in 12 orifice plates of sterilization cover glass in advance, and nutrient solution is the L-DMEM nutrient solutions containing 15%FBS, and 5~7d of culture is removed after not adherent cell, obtains attached cell；
(3) qualified donor material archives：The identification and analysis of purity and homogeneity is carried out to attached cell with reference to international mescenchymal stem cell standard, qualified mescenchymal stem cell is determined, sets up associated profiles, while being passed on to qualified stem cell, 4~5d is passed on once；
(4) mescenchymal stem cell into cartilage differentiation：By the subculture stem cell of more than 3 times, it is 4 × 10 that concentration, which is made,5/ ml suspension, 1ml cell suspensions are taken in 15m1 plastic centrifuge tubes, 500g centrifuges 15min, forms it into cell micelle, carefully sucks nutrient solution, add 2ml serum-free chondrocyte induction nutrient solutions, it is subsequently placed in CO2gas incubator and cultivates, changes liquid first after 4 days, changed liquid every 2 days later, the cell mass of culture is taken out after cultivating 21 days, cartilage differentiation effect is detected into；
(5) it is implanted into Regeneration and Repair effect：Qualified mescenchymal stem cell is implanted at patient articular by finite concentration, regeneration of joints repairing effect is observed.
2. method according to claim 1, wherein step (1) described preservation condition is aseptically, 4 DEG C of storages are less than 4 hours, are maintained in transportation under 4 DEG C of environment.
3. method according to claim 1, wherein walking cell culture environment condition described in poly- (2) and (4) for 37 DEG C, 5%CO2, saturated humidity CO2gas incubator in cultivate.
4. method according to claim 1, wherein walking serum-free chondrocyte induction nutrient solution described in poly- (4) refers to DMEM in high glucose (H-DMEM) 11ng/mL containing TGF-β, dexamethasone 10- 7Mmol/L, vitamin C 50mg/L, insulin 6.25ng/L, transferrins 6.25ug/mL, bovine serum albumin(BSA) 1.25ug/mL.
5. method according to claim 1, be implanted into described in poly- (5) concentration of stem cells 50~4 × 10 wherein walking7/ml。
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| CN108542916A (en) * | 2018-07-20 | 2018-09-18 | 深圳市第二人民医院 | It is a kind of to be used to treat arthral fluid mescenchymal stem cell preparation of Osteoarthritis and preparation method thereof |
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| WO2024144250A1 (en) * | 2022-12-29 | 2024-07-04 | 가톨릭관동대학교산학협력단 | Cartilage regeneration composition comprising synovial fluid-derived stem cells and chondrocytes |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106265739A (en) * | 2016-08-11 | 2017-01-04 | 李俊 | A kind of method utilizing arthroscope flushing liquor to prepare mescenchymal stem cell preparation |
| WO2018032123A1 (en) * | 2016-08-17 | 2018-02-22 | 李俊 | Method for preparing mesenchymal stem cell preparation by using arthroscopic irrigation solution |
| CN108542916A (en) * | 2018-07-20 | 2018-09-18 | 深圳市第二人民医院 | It is a kind of to be used to treat arthral fluid mescenchymal stem cell preparation of Osteoarthritis and preparation method thereof |
| CN108542917A (en) * | 2018-07-20 | 2018-09-18 | 深圳市第二人民医院 | A kind for the treatment of rheumatic ostealgia disease treatment injection and preparation method thereof with person joint's liquid mescenchymal stem cell excretion body extract |
| WO2024144250A1 (en) * | 2022-12-29 | 2024-07-04 | 가톨릭관동대학교산학협력단 | Cartilage regeneration composition comprising synovial fluid-derived stem cells and chondrocytes |
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