CN105748519A - Preparation method of adipose mesenchymal stem cell preparation for treating osteoarthritis - Google Patents
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Abstract
The invention relates to the field of treatment of osteoarthritis, and in particular relates to a preparation method of an adipose mesenchymal stem cell preparation for treating osteoarthritis. The method comprises: step 1, preparing microencapsulated adipose mesenchymal stem cells; step 2, preparing animal origin cartilage cells and synovial cells; step 3, performing inflammatory induction on the adipose mesenchymal stem cells; and step 4, treating the adipose mesenchymal stem cells. According to the preparation method of the adipose mesenchymal stem cell preparation for treating osteoarthritis, provided by the invention, the adipose mesenchymal stem cell preparation for treating osteoarthritis origin cells and the adipose mesenchymal stem cells are co-cultured, and a new in vitro co-culture system is invented, so that the adipose mesenchymal stem cells can obtain treatment characteristics for osteoarthritis outside a body, and can significantly promote the treatment effect on the osteoarthritis after being transplanted in to a body.
Description
Technical field
The present invention relates to osteoarthritis treatment field, the preparation method of especially a kind of fat mesenchymal stem cell preparation for treating osteoarthritis.
Background technology
Osteoarthritis is a kind of chronic joint diseases being chief complaint with Articular cartilage degeneration and Secondary cases hyperosteogeny, it is apt to occur in the positions such as the bigger knee joint of heavy burden, hip joint, spinal column and finger, have a strong impact on limb function and the daily life of patient, the person of being in a bad way there will be arthralgia, limitation of activity, possibly even cause joint deformity, even disabled, it is affect one of modal joint diseases of human health.Osteoarthritis sickness rate is very high, is the 4th disease affecting women's health, is the 8th disease affecting men's health.Less than 45 years old crowd's osteoarthritis prevalence is about 5%～10% according to statistics；But 55～65 years old, sickness rate was 40%～60%；And more than 75 years old crowd is 80%.Estimate that whole world Human Osteoarthritis total number of persons is up to more than 3.5 hundred million.Asia on average every 6 people just there is 1 people to suffer from osteoarthritis.
Osteoarthritis is as the typical Senile disease of one, and its pathogenesis is not still very clear and definite so far, increases as intra-osteal pressure, cytokine, matrix metalloproteinase, free radical, the factor such as immunoreation all can cause osteoarthritis.Existing treatment osteoarthritis mode mainly has naturopathy, Drug therapy, three kinds of modes of operative treatment.Naturopathy refers mainly to moderate exercise.The motion of suitable intensity can strengthen body function, thus slowing down the deterioration of osteoarthritis.Physiotherapy also includes utilizing sound, optical, electrical equipment to carry out ultrasound therapy, electromagnetotherapy, laser therapy, electrical stimulating therapy etc..These therapies more or less can reduce the secretion of joint II Collagenase Type, stimulates cellular metabolism, and easing the pain to wait acts on.But physical therapeutic effect is very limited, only as the remedy measures of a kind of auxiliary.
Drug therapy is the treatment means that current osteoarthritis is main, Drug therapy can be divided into again (1) non-steroidal drug, mainly includes aspirin, indomethacin, ibuprofen etc., and this kind of medicine effect is analgesia, but there is no the ability of antiinflammatory, it is impossible to stop the progress of the course of disease.And the non-steroidal drug of a new generation such as celecoxib, rofecoxib etc., namely there is the effect of antiinflammatory, it may have the effect of pain relieving.(2) medicine in nutrition joint, if such drug main nutrition protection cartilage structure, representational medicine has chondroitin sulfate, glucosamine sulfate, hyaluronic acid etc..(3) anti-oxidation medicine, Main Function is to resist produced reactive oxygen species and free radicals in elimination cellular process, protects the structure of the extracellular matrixs such as the hyaluronic acid in cartilaginous tissue, proteoglycan, collagen not to be destroyed.(4) anti-inflammatory drug, is primarily referred to as glucocorticoid, and this medicine of direct injection has remarkable effect for alleviating to have an intense pain, but this medicine can accelerate cartilage disease damage, therefore can not frequent application.
And when patients symptomatic is very serious, invalid through Drug therapy, have a strong impact on the patient of daily life, it may be considered that carry out operative treatment.Current therapeutic method of surgery is various, and every kind of method indication is different, and joint debridement art, tibial tubercle vancement are only used for the pathology that the state of an illness is smaller, and for arthropathy patient heavily and widely, TKA is final selection.
Relative to three of the above therapeutic scheme, stem-cell therapy is a kind of new osteoarthritis therapeutic modality risen gradually recently, especially mesenchymal stem cells.Mescenchymal stem cell is the cell that a class has self renewal and Multidirectional Differentiation ability, it is possible to be divided into the Various Tissues such as bone, cartilage, fat.It is low that mescenchymal stem cell also has immunogenicity, regulates organism immune response, the features such as cytokine secretion ability is strong, has the potentiality for the treatment of osteoarthritis.
Fat mesenchymal stem cell is the important kind in mescenchymal stem cell, fat mesenchymal stem cell not only possesses all of biological nature of mescenchymal stem cell, also having (1) to draw materials easily, because adipose tissue-derived is extensive, only patient need to be carried out local anesthesia can carry out fat absorption method；(2) in fat, stem cell population is more compared with in bone marrow；(3) fat mesenchymal stem cell in-vitro multiplication speed is higher.Substantial amounts of preclinical study has been had to confirm at present, fat mesenchymal stem cell can treat osteoarthritis, and no matter it is local joint injection, or joint cavity injection, all achieve good effect, fat mesenchymal stem cell has not only delayed arthritic further development, promotes the formation of newborn hyaline cartilage, and does not have toxic and side effects.Therefore the clinical experiment of fat mesenchymal stem cell treatment osteoarthritis is also carried out at country variant successively, and all achieves positive clinical therapeutic efficacy.Although mescenchymal stem cell treatment articular cartilage makes great progress, the therapeutic effect of mescenchymal stem cell is also unstable, and curative effect is also required to further raising.
Different organizational environments is had different responses by mescenchymal stem cell, when mescenchymal stem cell is in osteoarthritis environment, the state of own cells can be adjusted, secretion is suitable to improve arthritic cytokine or related nutritional molecule, can suddenly in the face of strong inflammation and free radical environment but be transplanted to suddenly intraarticular when mescenchymal stem cell, cytoactive and function are affected, thus reducing the therapeutic effect of osteoarthritis.
Summary of the invention
The preparation method that the invention aims to solve the deficiency of above-mentioned technology and provide a kind of fat mesenchymal stem cell preparation for treating osteoarthritis, it utilizes the cell that osteoarthritis is originated to co-culture with fat mesenchymal stem cell in vitro, make fat mesenchymal stem cell be obtained with the treatment characteristic for osteoarthritis in vitro, after in transplant, the therapeutic effect to osteoarthritis can be obviously improved.
In order to achieve the above object, the preparation method of the fat mesenchymal stem cell preparation for treating osteoarthritis designed by the present invention, the first step, the preparation of microencapsulation fat mesenchymal stem cell: collect the fat mesenchymal stem cell cultivating 3 generations in generation-10 terminated, fat mesenchymal stem cell is abundant, it is suspended in sodium alginate uniformly, carboxymethyl chitosan, in the solution of three kinds of material mixed preparing of PEG-4000, the wherein concentration of three kinds of materials respectively 1%～3%, 1%～3%, 4～10%, its base solvent is normal saline, the final concentration that fat mesenchymal stem cell is suspended in mixed liquor controls 1 × 105～1 × 106, the mixed liquor containing fat mesenchymal stem cell is joined in microsyringe, is then injected into the CaCl containing 40～100mmol/L2DMEM culture fluid in, the liquid pearl sphere diameter controlling liquid is not more than 0.5mm, by the liquid pearl of fatty mescenchymal stem cell containing CaCl2Liquid in stand 30 seconds～1 minute formed gel micro-ball, then gel micro-ball is forwarded in DMEM culture medium stand-by；
Having the method comprises the steps of firstly, preparing the gel micro-ball of a kind of fatty mescenchymal stem cell, this gel micro-ball is mainly prepared from by sodium alginate, carboxymethyl chitosan, three kinds of materials of PEG-4000.Sodium alginate is anionic compound, containing Ca2+Solution in can form gel, but sodium alginate gel stability in electrolyte solution is excessively poor.Carboxymethyl chitosan is to be cation also anion-containing polysaccharide, and carboxymethyl chitosan can be obviously improved sodium alginate gel stability in electrolyte solution together with sodium alginate.Simultaneously Polyethylene Glycol can improve the anti-degradation property of sodium alginate gel microsphere, it is to avoid gel micro-ball with fat mesenchymal stem cell, and when the synovial cell related to below, chondrocyte etc. co-culture, structure is not destroyed by the enzyme of emiocytosis or compound.The simultaneously permeability that also can improve sodium alginate gel microsphere of PEG-4000, makes gel micro-ball be easier to carry out the exchange of material with the external world.
Second step, prepare chondrocyte and the synovial cell of animal origin: prepare the mammal that 6～December is big, utilize the mixed enzyme solution of normal saline papain and collagenase, the concentration of papain is 2%～6%, wherein collagenase adopts II Collagenase Type, the concentration of II Collagenase Type is 1%～3%, mixed enzyme solution was injected in the mammiferous hip joint of 0.2～0.5mL to every every 2～3 days, injection 3～6 times altogether, after two weeks after having injected to 2 months, open hip joint, synovial tissue and cartilaginous tissue is taken out when visible joint cartilage has an obvious degeneration；
The main purpose of this step is to prepare mammiferous osteoarthritis, enzyme injection is the preparation method of a kind of conventional two kind osteoarthritis animal models, papain is a kind of hydrolytic enzyme, II Collagenase Type can directly act on cartilage matrix, when, after two kinds of enzyme combined effect articular cartilage, articular cartilage surface erosion, burn into necrocytosis being caused, and cause that subchondral bony sclerosis and bone cyst and hyperosteogeny are formed, and thicken with synovitis, joint, chondrocyte fall into disarray.Very much like with the clinical pattern of osteoarthritis.
3rd step, struvite induction to fat mesenchymal stem cell: after synovial tissue is taken out with cartilaginous tissue, aseptic balance liquid is utilized to clean multipass, by piece of tissue into about the small volume for 1mm × 1mm × 1mm of synovial tissue and cartilaginous tissue mosquito forceps and scissor cut, piece of tissue is put in culture bottle, pouring a small amount of DMEM culture fluid containing 10% hyclone into, culture fluid slightly covers piece of tissue surface, at 37 DEG C, 5%CO2Concentration, cultivate 4～6 hours under 95% damp condition, it is subsequently adding enough α～MEM culture fluid, after cultivating 3～15 days, change DMEM culture fluid and go not adherent cell and piece of tissue, gel micro-ball containing mescenchymal stem cell is joined in above-mentioned culture bottle simultaneously, with synovial tissue, cartilaginous tissue co-cultures, after cultivating 3～7 days, the gel micro-ball of fatty mescenchymal stem cell filtered to obtain by the screen cloth utilizing 100～300 orders, aseptic balance solution is utilized to clean gel micro-ball 5～10 times, wherein the culture fluid composition of co-culture system is: base solvent is DMEM, containing 10% hyclone, the il-1 of 1～3ng/mL, the interleukin-17 of 1～3ng/mL, the tumor necrosis factor of 1～3ng/mL；
Mescenchymal stem cell is in different organizational environment activity in mid-term, effect can be variant, in utilizing fat mesenchymal stem cell treatment osteoarthritis, fat mesenchymal stem cell is mainly by the effect of paracrine, nutrition chondrocyte and the degeneration for chondrocyte provide protection with damage, but after normal MSC injects joint, due to adapt to tissue in pathological changes environment and produce biological effect needs some cycles, but fat mesenchymal stem cell survival rate in the inverse ring borders such as arthritis is originally just relatively low, therefore the synovial cell of fat mesenchymal stem cell with arthritis source is co-cultured by the present invention with chondrocyte, suitable arthritis environment is provided to MSC, and utilize gel micro-ball the cell separation of a fat mesenchymal stem cell Yu animal origin to be opened, do not result in cross-contamination.The Transwell co-culture of cells method that the method is more traditional, is more easy to and carries out large-scale culture, be also easy to industrialization and prepare.Co-culture system natively adds a certain amount of inflammatory factor in addition, it is possible to adapt to in advance mono-inverse ring border of MSC, to ensure that cell injects postarticular biological activity, promoted MSC and treat the ability of osteoarthritis.
null4th step，The process of fat mesenchymal stem cell: the gel micro-ball of the fat mesenchymal stem cell cultivated after terminating is added containing 0.05%～0.1% disodiumedetate、In the DMEM culture fluid of 1%～5% trisodium citrate，Incubation 1～3 hour under 37 DEG C of conditions，Until gel micro-ball structural collapse，Soft method is adopted to be broken up fully in a liquid by fat mesenchymal stem cell，Until invisible gel structure，Then it is filtered with the screen cloth of 100～300 orders，Collect the suspension of fat mesenchymal stem cell，It is then centrifuged for collecting fat mesenchymal stem cell，Aseptic balance solution is utilized to be carried out 3～6 times，Trypan blue staining is utilized to carry out the detection of cell viability before using，When fat mesenchymal stem cell motility rate is more than 80%，After fat mesenchymal stem cell and joint injection hyaluronic acid solution are sufficiently mixed，It is 5 × 10 to cell concentration6～5 × 107；This fat mesenchymal stem cell suspension is fat mesenchymal stem cell preparation.
This step adopts disodiumedetate and trisodium citrate to be all metal ion chelation agents, because what gel structure prepared by the present invention relied primarily on is the effect of metal ion crosslinked, the metal ion chelation agent nontoxic, safe hence with both can promote the gel structure disintegrate of microsphere, such that it is able to cell is taken out again from gel structure, for the cell therapy of osteoarthritis.
Finally give above-mentioned fat mesenchymal stem cell preparation and carry out intra-articular injection treatment osteoarthritis.
The preparation method of the fat mesenchymal stem cell preparation for treating osteoarthritis that the present invention is obtained, the cell that osteoarthritis is originated is utilized to co-culture with fat mesenchymal stem cell in vitro, and invented a kind of new co culture system in vitro system, make fat mesenchymal stem cell be obtained with the treatment characteristic for osteoarthritis in vitro, after in transplant, the therapeutic effect to osteoarthritis can be obviously improved.
Detailed description of the invention
The present invention is further described by the following embodiment.
Embodiment 1:
The preparation method of the fat mesenchymal stem cell preparation for treating osteoarthritis that the present embodiment describes, concretely comprises the following steps: 1, the preparation of arthritis animal model
(1) preparing rabbit age is the new zealand white rabbit of 6 months；
(2) weigh the papain of 0.2g, then weigh the II Collagenase Type of 0.2g, utilize the normal saline of 10mL by after 2 kinds of enzymes mixing, and fully dissolve；
(3) mixed enzyme solution to the injection of hip joint 0.3mL of new zealand white rabbit, every injection in 2 days once, injection 3 times altogether；
(4), after normal condition is raised 2 months, pentobarbital sodium injecting anesthetic new zealand rabbit, hip joint is opened in operation, isolates synovial tissue of joint and cartilaginous tissue.
2, fat mesenchymal stem cell is prepared
(1) take subcutaneus adipose tissue to be about 20g and be dipped in DMEM basal medium, under hundred grades of conditions, utilize phosphate buffer to rinse fatty tissue, and remove the fiber in fatty tissue and vascular components, physical shear with smash to pieces；
(2) add 1% collagenase solution of 2 times of volumes, under 37 DEG C of conditions, digest fatty tissue 1 hour；
(3) utilizing the DMEM culture fluid containing 10% hyclone directly to terminate digestion, after 200 mesh filter screens filter out indigested piece of tissue, 800g collects cell in centrifugal 5 minutes；
(4) cell collected, and with the DMEM of 10% hyclone resuspended after, with 2 × 105Amount be inoculated in the culture bottle of 50mL, hatch in incubator cultivation change liquid every other day, until cell confluency reaches 80%；
(5) being digested latter 30 seconds by the cell EDTA of 0.25% trypsin and 0.03%, resuspended inoculation, with the form of 1 biography 2, cell is carried out Secondary Culture, culture fluid still adopts the DMEM of 10% hyclone；
(6) when cell confluency reaches to continue after 80% to go down to posterity with the form of 1 biography 2, until the 5th generation.
3, fatty mescenchymal stem cell gel micro-ball is prepared
(1) sodium alginate of 1.5g is weighed, the carboxymethyl chitosan of 1g, the PEG-4000 of 8g, utilize normal saline quantitatively to arrive 100g；
(2) by the 5th well-mixed gel rubber system of fat subsitutes mescenchymal stem cell just cultivating end, cell concentration controls 5 × 105；
(3) syringe of 500 μ l is utilized, by gel injection to containing 80mmol/LCaCl2DMEM cultivate in, stand about 1 minute；
(4) utilize the screen cloth of 200 orders, collect by filtration gel micro-ball, be then immersed in DMEM culture fluid stand-by.
4, co-culture of cells
(1) by taking out after synovial tissue and cartilaginous tissue be about 20g from the joint of animal of arthritis model, clean 3 times with phosphate buffer, then with instruments such as mosquito forceps and shears, piece of tissue is cut into the small tissue blocks lower than 1mm × 1mm × 1mm；
(2) small tissue blocks is put in the culture bottle of 2 75mL, pour a small amount of DMEM culture fluid containing 10% hyclone into, it is ensured that culture fluid slightly covers piece of tissue surface；
(3) at 37 DEG C, 5%CO2Under concentration, 95% damp condition after cultivation 6 as a child, it is subsequently adding the DMEM culture fluid containing 10% hyclone of 20mL, after cultivating 3 days, changes the new DMEM culture fluid containing 10% hyclone of 20mL, and go not adherent cell and piece of tissue；
(4) joining in cultivating system by the gel micro-ball containing mescenchymal stem cell, the cell concentration that every individual system adds is about 1 × 106, synovial cell, the chondrocyte originated with primary osteoarthritis co-culture, co-culture culture fluid used be containing 10% hyclone, the il-1 of 2ng/mL, the interleukin-17 of 2ng/mL, 2ng/mL the DMEM culture fluid of tumor necrosis factor；
(5) culture fluid more renewed every day, cultivates 5 days.
5, fat mesenchymal stem cell prepares and injection
(1) weigh 50mg disodiumedetate and 3g trisodium citrate, utilize solvent based on the DMEM culture medium of 100mL fully it fully to be dissolved；
(2) by co-culturing the microsphere of the fatty mescenchymal stem cell after end, pour in solution, incubation 1 hour under 37 DEG C of conditions；
(3) solution is then blown and beaten gently with pipet, until without being evident that gel structure；
(4) liquid is transferred in centrifuge tube, centrifugal 5 minutes of 800G；
(5) outwell supernatant fluid, utilize the resuspended cleaning cell number time of phosphate buffer；
(6) taking the ratio of a small amount of cell cell tests instrument detection living cells, living cells rate requirement is more than more than 80%；
(7) then fat mesenchymal stem cell and injection hyaluronic acid solution are sufficiently mixed, are 1 × 10 to cell concentration7；Namely this fat mesenchymal stem cell suspension is fat mesenchymal stem cell preparation.
(8) carry out joint injection, treat osteoarthritis.
Embodiment 2:
The preparation method of the fat mesenchymal stem cell preparation for treating osteoarthritis that the present embodiment describes, concretely comprises the following steps: 1, the preparation of arthritis animal model
(1) preparing rabbit age is the new zealand white rabbit of 10 months；
(2) weigh the papain of 0.4g, then weigh the II Collagenase Type of 0.2g, utilize the normal saline of 10mL by after 2 kinds of enzymes mixing, and fully dissolve；
(3) mixed enzyme solution to the injection of hip joint 0.5mL of new zealand white rabbit, every injection in 2 days once, injection 6 times altogether；
(4), after normal condition is raised 2 months, pentobarbital sodium injecting anesthetic new zealand rabbit, hip joint is opened in operation, isolates synovial tissue of joint and cartilaginous tissue.
2, fat mesenchymal stem cell is prepared
(1) taking subcutaneus adipose tissue to be about 25g and be dipped in DMEM basal medium, under super-clean bench, PBS rinses fatty tissue, and removes the fiber in fat and vascular components, physical shear with smash to pieces；
(2) add 1% collagenase solution of 2 times of volumes, under 37 DEG C of conditions, digest fatty tissue 3 hours；Period constantly rocks facilitating digestion.
(3) utilizing the DMEM culture fluid containing 10% hyclone directly to terminate digestion, after 200 mesh filter screens filter out indigested piece of tissue, 800g collects cell in centrifugal 5 minutes；
(4) cell collected, and with the DMEM of 10% hyclone resuspended after, with 2 × 105Amount be inoculated in the culture bottle of 50mL, hatch in incubator cultivation change liquid every other day, until cell confluency reaches 80%；
(5) being digested latter 30 seconds by the cell EDTA of 0.25% trypsin and 0.03%, resuspended inoculation, with the form of 1 biography 2, cell is carried out Secondary Culture, culture fluid still adopts the DMEM of 10% hyclone；
(6) when cell confluency reaches to continue after 80% to go down to posterity with the form of 1 biography 2, until the 3rd generation.
3, fatty mescenchymal stem cell gel micro-ball is prepared
(1) sodium alginate of 1.5g is weighed, the carboxymethyl chitosan of 1g, the PEG-4000 of 8g, utilize normal saline quantitatively to arrive 100g；
(2) by the 3rd well-mixed gel rubber system of fat subsitutes mescenchymal stem cell just cultivating end, cell concentration controls 0.5 × 106；
(3) syringe of 500 μ l is utilized, by gel injection to containing 80mmol/LCaCl2DMEM cultivate in, stand about 1 minute；
(4) utilize the screen cloth of 300 orders, collect by filtration gel micro-ball, be then immersed in DMEM culture fluid stand-by.
4, co-culture of cells
(1) by taking out after synovial tissue and cartilaginous tissue be about 20g from the joint of animal of arthritis model, clean 3 times with phosphate buffer, then with instruments such as mosquito forceps and shears, piece of tissue is cut into the small tissue blocks lower than 1mm × 1mm × 1mm；
(2) small tissue blocks is put in the culture bottle of 2 75mL, pour a small amount of DMEM culture fluid containing 10% hyclone into, it is ensured that culture fluid slightly covers piece of tissue surface；
(3) at 37 DEG C, 5%CO2Under concentration, 95% damp condition after cultivation 6 as a child, it is subsequently adding the DMEM culture fluid containing 10% hyclone of 20mL, after cultivating 3 days, changes the new DMEM culture fluid containing 10% hyclone of 20mL, and go not adherent cell and piece of tissue；
(4) joining in cultivating system by the gel micro-ball containing mescenchymal stem cell, the cell concentration that every individual system adds is about 1 × 106, synovial cell, the chondrocyte originated with primary osteoarthritis co-culture, co-culture culture fluid used be containing 10% hyclone, the il-1 of 3ng/mL, the interleukin-17 of 3ng/mL, 3ng/mL the DMEM culture fluid of tumor necrosis factor；
(5) culture fluid more renewed every day, cultivates 7 days.
5, fat mesenchymal stem cell prepares and injection
(1) weigh 80mg disodiumedetate and 5g trisodium citrate, utilize solvent based on the DMEM culture medium of 100mL fully it fully to be dissolved；
(2) by co-culturing the microsphere of the fatty mescenchymal stem cell after end, pour in solution, incubation 3 hours under 37 DEG C of conditions；
(3) solution is then blown and beaten gently with pipet, until without being evident that gel structure；
(4) liquid is transferred in centrifuge tube, centrifugal 5 minutes of 800G；
(5) outwell supernatant fluid, utilize the resuspended cleaning cell number time of phosphate buffer；
(6) taking the ratio of a small amount of cell cell tests instrument detection living cells, living cells rate requirement is more than more than 80%；
(7) then fat mesenchymal stem cell and injection hyaluronic acid solution are sufficiently mixed, are 2 × 10 to cell concentration7；Namely this fat mesenchymal stem cell suspension is fat mesenchymal stem cell preparation.
(8) carry out joint injection, treat osteoarthritis.
Claims (2)
1. the preparation method for treating the fat mesenchymal stem cell preparation of osteoarthritis, it is characterized in that: the first step, the preparation of microencapsulation fat mesenchymal stem cell: collect the fat mesenchymal stem cell cultivating 3 generations in generation-10 terminated, fat mesenchymal stem cell is abundant, it is suspended in sodium alginate uniformly, carboxymethyl chitosan, in the solution of three kinds of material mixed preparing of PEG-4000, the wherein concentration of three kinds of materials respectively 1%～3%, 1%～3%, 4～10%, its base solvent is normal saline, the final concentration that fat mesenchymal stem cell is suspended in mixed liquor controls 1 × 105～1 × 106, the mixed liquor containing fat mesenchymal stem cell is joined in microsyringe, is then injected into the CaCl containing 40～100mmol/L2DMEM culture fluid in, the liquid pearl sphere diameter controlling liquid is not more than 0.5mm, by the liquid pearl of fatty mescenchymal stem cell containing CaCl2Liquid in stand 30 seconds～1 minute formed gel micro-ball, then gel micro-ball is forwarded in DMEM culture medium stand-by；Second step, prepare chondrocyte and the synovial cell of animal origin: prepare the mammal that 6～December is big, utilize the mixed enzyme solution of normal saline papain and collagenase, the concentration of papain is 2%～6%, wherein collagenase adopts II Collagenase Type, the concentration of II Collagenase Type is 1%～3%, mixed enzyme solution was injected in the mammiferous hip joint of 0.2～0.5mL to every every 2～3 days, injection 3～6 times altogether, after two weeks after having injected to 2 months, open hip joint, synovial tissue and cartilaginous tissue is taken out when visible joint cartilage has an obvious degeneration；3rd step, struvite induction to fat mesenchymal stem cell: after synovial tissue is taken out with cartilaginous tissue, aseptic balance liquid is utilized to clean multipass, synovial tissue and cartilaginous tissue are cut into the small tissue blocks of volume comparatively 1mm × 1mm × 1mm, piece of tissue is put in culture bottle, pouring a small amount of DMEM culture fluid containing 10% hyclone into, culture fluid slightly covers piece of tissue surface, at 37 DEG C, 5%CO2Concentration, cultivate 4～6 hours under 95% damp condition, it is subsequently adding enough α～MEM culture fluid, after cultivating 3～15 days, change DMEM culture fluid and go not adherent cell and piece of tissue, gel micro-ball containing mescenchymal stem cell is joined in above-mentioned culture bottle simultaneously, with synovial tissue, cartilaginous tissue co-cultures, after cultivating 3～7 days, the gel micro-ball of fatty mescenchymal stem cell filtered to obtain by the screen cloth utilizing 100～300 orders, aseptic balance solution is utilized to clean gel micro-ball 5～10 times, wherein the culture fluid composition of co-culture system is: base solvent is DMEM, containing 10% hyclone, the il-1 of 1～3ng/mL, the interleukin-17 of 1～3ng/mL, the tumor necrosis factor of 1～3ng/mL；null4th step，The process of fat mesenchymal stem cell: the gel micro-ball of the fat mesenchymal stem cell cultivated after terminating is added containing 0.05%～0.1% disodiumedetate、In the DMEM culture fluid of 1%～5% trisodium citrate，Incubation 1～3 hour under 37 DEG C of conditions，Until gel micro-ball structural collapse，Soft method is adopted to be broken up fully in a liquid by fat mesenchymal stem cell，Until invisible gel structure，Then it is filtered with the screen cloth of 100～300 orders，Collect the suspension of fat mesenchymal stem cell，It is then centrifuged for collecting fat mesenchymal stem cell，Aseptic balance solution is utilized to be carried out 3～6 times，Trypan Blue is utilized to carry out the detection of cell viability before using，When fat mesenchymal stem cell motility rate is more than 80%，After fat mesenchymal stem cell and joint injection hyaluronic acid solution are sufficiently mixed，It is 5 × 10 to cell concentration6～5 × 107；This fat mesenchymal stem cell suspension is fat mesenchymal stem cell preparation.
2. the preparation method of a kind of fat mesenchymal stem cell preparation for treating osteoarthritis according to claim 1, it is characterized in that: synovial tissue and cartilaginous tissue are cut into the piece of tissue of small volume by second step, wherein piece of tissue be sized to 1mm × 1mm × 1mm or less than 1mm × 1mm × 1mm.
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| CN106333965A (en) * | 2016-10-31 | 2017-01-18 | 遵义医学院附属医院 | Preparation for treating osteoarthritis and treatment method |
| 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 |
| CN108815509A (en) * | 2018-08-07 | 2018-11-16 | 天津昂赛细胞基因工程有限公司 | Mescenchymal stem cell substitution product and its application comprising degradable biological medical slow-release material |
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| CN109251887A (en) * | 2018-10-12 | 2019-01-22 | 山东麦德克斯生物科技有限公司 | A kind of preparation method and cartilaginous tissue of cartilage cell's primer layer |
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| CN114015648A (en) * | 2021-10-20 | 2022-02-08 | 成都拜美森生物科技有限公司 | High-performance adipose-derived mesenchymal stem cell solution and preparation method and application thereof |
| CN114404370A (en) * | 2021-12-13 | 2022-04-29 | 苏州市立医院 | Nano-fat functionalized injectable super-lubricating microsphere and preparation method and application thereof |
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| CN106267353A (en) * | 2016-09-19 | 2017-01-04 | 广州市拜沃思生物科技有限公司 | A kind of fat stem cell filler and preparation method thereof |
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| CN111607560A (en) * | 2019-02-22 | 2020-09-01 | 深圳安吉赛尔生物科技有限公司 | Culture method of adipose-derived stem cells and application of adipose-derived stem cells in arthritis |
| CN114015648A (en) * | 2021-10-20 | 2022-02-08 | 成都拜美森生物科技有限公司 | High-performance adipose-derived mesenchymal stem cell solution and preparation method and application thereof |
| CN114015648B (en) * | 2021-10-20 | 2024-02-20 | 成都拜美森生物科技有限公司 | High-performance adipose-derived mesenchymal stem cell solution and preparation method and application thereof |
| CN114404370A (en) * | 2021-12-13 | 2022-04-29 | 苏州市立医院 | Nano-fat functionalized injectable super-lubricating microsphere and preparation method and application thereof |
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| CN114469899A (en) * | 2022-01-26 | 2022-05-13 | 广州喜舍俪健康管理咨询有限公司 | Preparation method of stem cell factor sustained-release microspheres for repairing endometrial injury |
| CN116115661A (en) * | 2022-12-20 | 2023-05-16 | 湖南华启细胞生物科技有限公司 | Adipose-derived mesenchymal stem cell preparation for treating joint degenerative disease and preparation method thereof |
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