CN104988117A - Method for separating and culturing mesenchymal stem cells from umbilical cord and inducing and differentiating mesenchymal stem cells into cartilage cells - Google Patents
Method for separating and culturing mesenchymal stem cells from umbilical cord and inducing and differentiating mesenchymal stem cells into cartilage cells Download PDFInfo
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- CN104988117A CN104988117A CN201510386692.2A CN201510386692A CN104988117A CN 104988117 A CN104988117 A CN 104988117A CN 201510386692 A CN201510386692 A CN 201510386692A CN 104988117 A CN104988117 A CN 104988117A
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Abstract
The invention provides a method for separating and culturing mesenchymal stem cells from an umbilical cord and inducing and differentiating the mesenchymal stem cells into cartilage cells. The method comprises the steps that the umbilical cord is treated; the cells are separated from the umbilical cord and umbilical vessels and subjected to primary cell culture; cell subculture is conducted, and the mesenchymal stem cells are obtained; the mesenchymal stem cells are induced and differentiated into the cartilage cells, wherein in the step that the cells are separated from the umbilical cord and the umbilical vessels, the umbilical cord and the umbilical vessels are digested through digestive juice. By means of the method, the mesenchymal stem cells can be separated from the umbilical cord and cultured, and the obtained mesenchymal stem cells are subjected to further induced transformation; the method can be used for mass production of the mesenchymal stem cells and has importance significance in treatment of injured tissue which can hardly regenerate.
Description
[technical field]
The present invention relates to mescenchymal stem cell technical field, particularly relate to and be a kind ofly separated from umbilical cord and cultivate mescenchymal stem cell and to the method for chondrogenic differentiation.
[background technology]
Osteoarthritis (Osteoarthritis, OA) be referred to as " not dead cancer ", it is a kind of chronic progressive external bone and joint diseases being pathological characters with cartilage degeneration and periarticular hyperosteogeny that incidence rises with age growth, main infringement joint cartilage, bone and synovial tissue, cause arthralgia, deformity and dysfunction, be the most common cause causing middle-aged and old pain, and this disease have certain disability rate, has had a strong impact on the quality of life of patient.The treatment of current osteoarthritis mainly comprises: motion and Life Guidance, Physiotherapy, (non-steroidal anti-inflammatory drugs, glucosamine are oral in pharmacological agent, local injection hyaluronic acid sodium and hormone etc.), surgical arthroscopic art etc., improve the quality of life of patient to a certain extent, but still there is no desirable methods for the treatment of, in the urgent need to seeking new methods for the treatment of and technology.This disease finally can cause the damage of joint cartilage, if can repair impaired cartilage obviously will improve conditions of patients, therefore, how repairing impaired cartilage and keep its function, is the therapeutic goal of osteoarthritis.But being difficult to regeneration due to articular chondrocytes and repairing, is the bottleneck that restriction osteoarthritis treatment further develops.
Utilize differentiation of stem cells for chondrocyte, it is the new methods for the treatment of impelling articular chondrocytes to regenerate and repair, this methods for the treatment of is planted by the stem cell that the function of vitro culture is correlated with in natural or synthetic, there is good biocompatibility, degradable and having on the polymer support of certain three-dimensional structure, thus define cytoskeleton mixture, then be implanted into tissue defect, thus formation joint cartilage, realize the renewable reservoir of cartilage.Wherein, mescenchymal stem cell (Mesenchymal Stem Cells, MSCs) is one of conventional cell.
Up to now, marrow is the main source of mescenchymal stem cell, but bone marrow collection is an invasive technique, sometimes puncture is needed to gather for several times, and need anaesthetize sb. generally during operation, surgical procedure is complicated and difficult, therefore brings serious mind & body burden to patient.
Umbilical cord is the cord structures of connection embryo umbilical region and the placenta part formed by trophocyte in embry ogenesis process, covers amnion outward, includes mucus reticular tissue.Wherein outside the yolk sac of oil removing locking in reticular tissue and urachus, also have a vein and two arteries.Umbilical cord is the passage of stem cell formation and process.Therefore, the mescenchymal stem cell of being originated by umbilical cord also has self-renewal capacity under suitable condition, therefore can carry out enlarged culturing in turning to specific cells.With derive from marrow, muscle is compared with the mescenchymal stem cell of skin histology, umbilical cord source mescenchymal stem cell there is more remarkable differentiation capability.In addition, the mescenchymal stem cell in umbilical cord source has following advantage compared with mesenchymal stem cells MSCs: umbilical cord aboundresources, donor (mother and baby) is had no adverse effects, to HLA (Human Leukocyte Antigen, human leucocyte antigen) requirement that is harmonious of distribution type reduces relatively, can expand donor scope etc.
As disclosed the method for separation and Culture mescenchymal stem cell from umbilical cord in patent CN 103266081A, it specifically discloses and umbilical cord is carried out digesting rear employing mescenchymal stem cell substratum cultivates, obtain the concrete grammar of mescenchymal stem cell, but the pick-up rate of the mescenchymal stem cell adopting this method to obtain is lower.In prior art, the cost of mescenchymal stem cell separation and ientification high, mescenchymal stem cell low conversion rate, complicated operation, cannot realize the scale operation of mescenchymal stem cell.
[summary of the invention]
For overcoming separation and Culture mescenchymal stem cell from umbilical cord at present, and be that chondrocyte's process cost is high, the problem of low conversion rate by mesenchyma stem cell differentiation induction, the invention provides and be a kind ofly separated from umbilical cord and cultivate mescenchymal stem cell and to the method for chondrogenic differentiation.
The invention provides and be a kind ofly separated from umbilical cord and cultivate mescenchymal stem cell and to the method for chondrogenic differentiation, it comprises umbilical cord process, from umbilical cord and umbilical blood vessels isolated cell and carry out primary cell culture, passage cultivates and obtains mescenchymal stem cell and be chondrocyte by mesenchyma stem cell differentiation induction;
From umbilical cord and umbilical blood vessels isolated cell comprise adopt Digestive system umbilical cord and umbilical blood vessels are digested, this Digestive system is after DMEM (Dulbecco minimumessential medium) substratum mixes with F12 substratum 1:1, then adds 0.5-1.5mg/ml collagenase, 4.5-5.5 μ g/ml Unidasa, 100u/ml penicillin and 100 μ g/ml Streptomycin sulphate mixed configuration and form;
Primary cell culture comprises employing cell culture fluid and cultivates, and this cell culture fluid is after DMEM substratum mixes with F12 substratum 1:1, then adds 100u/ml penicillin, 100 μ g/ml Streptomycin sulphates and 10% foetal calf serum;
Passage cultivate obtain mescenchymal stem cell be included in primary cell culture until cell grow to 70%-85% merge time, the cell culture fluid in the culture dish removing original cuiture drawn by dropper, after adopting PBS washing, 0.25% trypsin-EDTA solutions adding 5-10ml again digests, digestion also observation of cell at 37 DEG C, treat that cell body is tending towards becoming bowlder, the cell culture fluid adding equivalent immediately stops digestion; Cell is blown and beaten gently with suction pipe, until attached cell suspends, cell needed for centrifugal acquisition; Make single cell suspension with cell culture fluid, primary cell carries out Secondary Culture by 1:2 and obtains 1st generation passage cell; Make to use the same method repeat 2nd generation and the 3rd generation passage cell cultivation;
By mesenchyma stem cell differentiation induction for chondrocyte comprises the 3rd generation (P3) cell with 7 × 10
4/ ml-1 × 10
7/ ml is seeded in culture plate, the cover glass of preset sterilization in culture plate, is replaced by the Induction Transformation nutrient solution containing inducible factor, culture plate is placed in 5%CO after cultivating 24h
2, cultivate in saturated humidity 80%, 37 DEG C of incubators, change liquid 2-3 time weekly;
Wherein, this Induction Transformation nutrient solution comprises: containing the DMEM/F12 nutrient solution of 5% foetal calf serum, 10ng/ml transforming growth factor (Transforming Growth Factor, TGF), β I, 6-6.5 μ g/ml Regular Insulin, 98-100 μm ol/ml dexamethasone, 0.5-1.5 μm ol/ml Sodium.alpha.-ketopropionate and 6-6.5 μ g/ml Transferrins,iron complexes.
Preferably, in umbilical cord treating processes, adopt dual anti-normal saline washing umbilical cord outside surface, this dual anti-salt solution is the physiological saline containing 100u/ml penicillin and 100 μ g/ml Streptomycin sulphates; Umbilical cord treating processes comprises the sludged blood removing umbilical cord and umbilical blood vessels surface, and changes container, umbilical cord clean for surface treatment is placed in dual anti-salt solution and soaks 15-18min; Take out umbilical cord, change container, then repeat with dual anti-saline soak 2-4 time, each 15-18min; Change sterile chamber, umbilical cord segment, changes sterile chamber again, longitudinally cuts the amnion on umbilical cord surface along both sides open, longitudinally tears umbilical cord, after removing Umbilical artery and umbilical vein and other blood vessel extravasated blood, washs to salt water colorless limpid in dual anti-salt solution.
Preferably, isolated cell from umbilical cord and umbilical blood vessels also carries out primary cell culture and comprises and the umbilical cord and umbilical blood vessels that complete extravasated blood process being transferred to respectively in aseptic digestion vessel, is shredded by tissue by sterile scissors; Then the tissue shredded and PBS are mixed and centrifugal acquisition throw out, the Digestive system prepared is added in throw out, is transferred to after blowing afloat precipitation in another sterile petri dish, and put into 37 °, sterile culture case digestion 4-7h; By the solution digested and organize centrifugal rear acquisition throw out; Throw out cell culture fluid is resuspended, and be inoculated in 10cm culture dish, cell is now called primary cell, be placed in 37 DEG C, saturated humidity, containing 5%CO
2cultivate in incubator; 24h full dose changes liquid, and every 2-3 days change nutrient solution.
Preferably, above-mentioned Digestive system is after DMEM substratum mixes with F12 substratum 1:1, then adds 0.5-1mg/ml collagenase, 4.5-5 μ g/ml Unidasa, 100u/ml penicillin and 100 μ g/ml Streptomycin sulphate mixed configuration and form.
Preferably, passage cultivates acquisition mescenchymal stem cell also can in primary cell culture when cell grows to 70%-80% fusion, the cell culture fluid in the culture dish removing original cuiture drawn by dropper, and after adopting PBS to wash, then 0.25% trypsin-EDTA solutions adding 5ml digests; And the 3rd generation (P3) cell all right 2 × 10
5/ ml-1 × 10
6/ ml is seeded to the cultivation that the laggard mesenchymal stem cells in the ranks of culture plate is induced to differentiate into chondrocyte.
Preferably, this Digestive system also can be after DMEM substratum mixes with F12 substratum 1:1, then adds 1mg/ml collagenase, 5 μ g/ml Unidasas, 100u/ml penicillin and 100 μ g/ml Streptomycin sulphate mixed configuration and form.
Preferably, this Induction Transformation nutrient solution comprises: containing sodium β-glycerophosphate and the 0-1.5 μm of ol/ml vitamins D of the DMEM/F12 nutrient solution of 5% foetal calf serum and 10ng/ml transforming growth factor-beta I, 6-6.5 μ g/ml Regular Insulin, 98-100 μm ol/ml dexamethasone, 0.5-1.5 μm ol/ml Sodium.alpha.-ketopropionate, 6-6.5 μ g/ml Transferrins,iron complexes, 0-2 μm ol/ml
3.
Further preferably, this Induction Transformation nutrient solution comprises: containing sodium β-glycerophosphate and 0.5 μm of ol/ml vitamins D of the DMEM/F12 nutrient solution of 5% foetal calf serum and 10ng/ml transforming growth factor-beta I, 6.25 μ g/ml Regular Insulin, 98 μm of ol/ml dexamethasone, 1 μm of ol/ml Sodium.alpha.-ketopropionate, 6.25 μ g/ml Transferrins,iron complexess, 1.5 μm of ol/ml
3.
Preferably, this Induction Transformation nutrient solution comprises: containing sodium β-glycerophosphate and 0.5 μm of ol/ml vitamins D of the DMEM/F12 nutrient solution of 5% foetal calf serum and 10ng/ml transforming growth factor-beta I, 6.25 μ g/ml Regular Insulin, 99 μm of ol/ml dexamethasone, 1 μm of ol/ml Sodium.alpha.-ketopropionate, 6.25 μ g/ml Transferrins,iron complexess, 0.5 μm of ol/ml
3.
Preferably, this Induction Transformation nutrient solution comprises: containing the sodium β-glycerophosphate of the DMEM/F12 nutrient solution of 5% foetal calf serum and 10ng/ml transforming growth factor-beta I, 6.25 μ g/ml Regular Insulin, 98 μm of ol/ml dexamethasone, 1 μm of ol/ml Sodium.alpha.-ketopropionate, 6.25 μ g/ml Transferrins,iron complexess, 2 μm of ol/ml.
Relative to prior art, first be separated from umbilical cord in the present invention and cultivate mescenchymal stem cell, and the mescenchymal stem cell of acquisition is carried out further Induction Transformation, may be used for the scale operation of the mescenchymal stem cell of so-called pluripotent cell, be significant in the treatment of the damaged tissue of regeneration difficulty.
Secondly, adopt the method described in the present invention, the survival rate being separated and cultivating mescenchymal stem cell from the umbilical cord of same donor, to more than 98-99%, obtains the pick-up rate of mescenchymal stem cell far above adopting separation and Culture in prior art.
Finally, adopt the preparation method described in the present invention, the specific marker being separated and cultivating mescenchymal stem cell strongly expressed from umbilical cord comprises CD44, CD105, CD90 and CD73, and CD45, CD34, CD14, CD19 and HLA-DR expresses feminine gender, the immunophenotype of the mescenchymal stem cell adopting method separation and Culture of the present invention to obtain is identical with the immunophenotype of mescenchymal stem cell, this illustrates the feature by the cell obtained from umbilical cord separation and cultivation mescenchymal stem cell and to the method separation and Culture of chondrogenic differentiation described in the present invention with mescenchymal stem cell.
To sum up, the chondrocyte adopting method of the present invention to prepare can be used in the treatment of human body cartilage defect, and can be beneficial to the scale operation of mescenchymal stem cell, has larger application prospect.
[accompanying drawing explanation]
Fig. 1 a is nucleus magnetic resonance (MRI) the situation schematic diagram that in the present invention, case 1 treats front joint;
Fig. 1 b-1e adopts nucleus magnetic resonance (MRI) the situation schematic diagram carrying out treating posterior joint in conjunction with method described in experimental group 11 in the embodiment of the present invention 2 to case 1;
Fig. 2 a is nucleus magnetic resonance (MRI) the situation schematic diagram that in the present invention, case 1 treats front joint;
Fig. 2 b-2e adopts nucleus magnetic resonance (MRI) the situation schematic diagram carrying out treating posterior joint in conjunction with method described in experimental group 11 in the embodiment of the present invention 2 to case 1;
Fig. 3 a is nucleus magnetic resonance (MRI) the situation schematic diagram that in the present invention, case 2 treats front joint;
Fig. 3 b adopts nucleus magnetic resonance (MRI) the situation schematic diagram carrying out treating posterior joint in conjunction with method described in experimental group 14 in the embodiment of the present invention 2 to case 2;
Fig. 4 a is nucleus magnetic resonance (MRI) the situation schematic diagram that in the present invention, case 2 treats front joint;
Fig. 4 b adopts nucleus magnetic resonance (MRI) the situation schematic diagram carrying out treating posterior joint in conjunction with method described in experimental group 14 in the embodiment of the present invention 2 to case 2;
Fig. 5 a is nucleus magnetic resonance (MRI) the situation schematic diagram that in the present invention, case 3 treats front joint;
Fig. 5 b adopts nucleus magnetic resonance (MRI) the situation schematic diagram carrying out treating posterior joint in conjunction with method described in the embodiment of the present invention 2 experimental group 11 to case 3;
Fig. 6 a is nucleus magnetic resonance (MRI) the situation schematic diagram that in the present invention, case 3 treats front joint;
Fig. 6 b adopts nucleus magnetic resonance (MRI) the situation schematic diagram carrying out treating posterior joint in conjunction with method described in the embodiment of the present invention 2 experimental group 11 to case 3;
Fig. 7 a is nucleus magnetic resonance (MRI) the situation schematic diagram that in the present invention, case 4 treats front joint;
Fig. 7 b adopts nucleus magnetic resonance (MRI) the situation schematic diagram carrying out treating posterior joint in conjunction with method described in the embodiment of the present invention 2 experimental group 14 to case 4;
Fig. 8 a is nucleus magnetic resonance (MRI) the situation schematic diagram that in the present invention, case 4 treats front joint;
Fig. 8 b adopts nucleus magnetic resonance (MRI) the situation schematic diagram carrying out treating posterior joint in conjunction with method described in the embodiment of the present invention 2 experimental group 14 to case 4;
Fig. 9 a is nucleus magnetic resonance (MRI) the situation schematic diagram that in the present invention, case 4 treats front joint;
Fig. 9 b adopts nucleus magnetic resonance (MRI) the situation schematic diagram carrying out treating posterior joint in conjunction with method described in the embodiment of the present invention 2 experimental group 14 to case 4.
[embodiment]
In order to make object of the present invention, technical scheme and advantage are clearly understood, below in conjunction with accompanying drawing and embodiment, are further elaborated to the present invention.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
embodiment 1, from umbilical cord, to be separated and to cultivate mesenchyme according to method described in the present invention do
cell
Umbilical cord and Cord Blood-Derived: take from 24, healthy puerpera's umbilical cord, carry out effectively frozen.
experimental group 1:
Be separated from umbilical cord and cultivate mescenchymal stem cell, it is specifically separated, cultural method is as follows:
(1) umbilical cord process:
Select the umbilical cord of healthy puerpera, with dual anti-normal saline washing umbilical cord outside surface, remove the sludged blood on surface.Change container, umbilical cord clean for surface treatment is placed in dual anti-salt solution and soaks 15min; Take out umbilical cord, change container, then repeat with dual anti-saline soak twice, each 15min.Change sterile chamber, cut one section that 5cm is long, again change sterile chamber, longitudinally cut the amnion on umbilical cord surface along both sides open, longitudinally tear umbilical cord, remove Umbilical artery and umbilical vein, in case of cannot the person of removal, then cut the blood vessel of extravasated blood open, extravasated blood is removed clean, finally wash to salt water colorless limpid in dual anti-salt solution.The Umbilical artery removed and umbilical vein are cut open, remove extravasated blood, wash to salt water colorless limpid in dual anti-salt solution.
Wherein, described dual anti-salt solution is the physiological saline containing 100u/ml penicillin and 100 μ g/ml Streptomycin sulphates.
(2) cellular segregation and primary cell culture:
The umbilical cord handled well and umbilical blood vessels are transferred in aseptic digestion vessel respectively, by sterile scissors, tissue are shredded; Then the tissue shredded and PBS substratum are added in centrifuge tube, centrifugal 1000r/min under normal temperature, maintain 5min.Remove supernatant, the Digestive system prepared is added in precipitation, blows afloat precipitation, be transferred in another sterile petri dish with transfer pipet, put into 37 °, sterile culture case digestion 6h.
This Digestive system comprises after DMEM substratum mixes with F12 substratum 1:1, then adds collagenase, Unidasa, penicillin and Streptomycin sulphate mixed configuration and form;
The solution digested and tissue are transferred to centrifuge tube, 300g, centrifugal 5min, abandons supernatant.Precipitation cell culture fluid is resuspended, is inoculated in 10cm culture dish.Tagged tissue title, date and cell algebraically, cell is now called primary cell (P0), is placed in 37 DEG C, and saturated humidity 80% is cultivated containing in 5%CO2 incubator.24h full dose changes liquid, within every 2-3 days later, changes nutrient solution.
Wherein, described cell culture fluid is after DMEM substratum and F12 substratum 1:1 are configured to 80ml solution, then adds 100u/ml penicillin, 100 μ g/ml Streptomycin sulphates and 10% foetal calf serum.
(3) passage:
When cell grows to about 70% fusion, the cell culture fluid in the culture dish removing original cuiture drawn by dropper, adopts PBS (phosphate buffered saline buffer) to wash 2 times, to wash away residual cell nutrient solution.After washing, then 0.25% trypsin-EDTA solutions adding 5ml digests, and digests about 2min at 37 DEG C.
In cell dissociation process, whether examine under a microscope cell at any time from wall, cell cytoplasm bounces back, and cell body is tending towards becoming bowlder, adds the cell culture fluid termination digestion of equivalent containing 10% foetal calf serum immediately.Blow and beat cell gently with suction pipe, until attached cell suspends, draw cell suspension and move into 15ml centrifuge tube, centrifugal 1000r/min under normal temperature, and maintain centrifugal 5min.After centrifugal, abandon supernatant liquor, bottom is required cell.
Make single cell suspension with cell culture fluid, primary cell carries out Secondary Culture by 1:2.Tagged tissue title, date and cell algebraically, cell is now called 1st generation passage cell (P1).
Make to use the same method repeat 2nd generation and the 3rd generation passage cell cultivation.
In fact experimental group 1 parameter can carry out the adjustment in certain limit, and concrete setting range is as follows:
The single time that umbilical cord clean for surface treatment is placed in dual anti-salt solution is not limited to 15min, and it also can soak 15-16min or 16-17min or 17-18min, and can repeat with dual anti-saline soak 2-3 time or 3-4 time.
The above-mentioned umbilical cord tissue shredded adds after PBS solution washs, and add Digestive system in experimental group 1, and digest at 37 ° of temperature, digestion time is not limited to 6h, and it can be 4-7h, or is 4-6h further, also can be 5-6h or 4-5h.
Can when cell grows to about 70-80% fusion in above-mentioned primary cell culture process, carry out the operation of next step again, its fusion degree also can be 70-75% or 75%-80%, can be specially 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%.
The content of the collagenase in Digestive system can be adjusted to 0.5-1.5mg/ml, 0.5-1mg/ml and 1-1.5mg/ml, and the content of Unidasa yet can be 4.5-5.5 μ g/ml, 4.5-5 μ g/ml and 5-5.5 μ g/ml.
Except experimental group 1, applicant adopts the Digestive system of different parameters value and 0.25 trypsin-EDTA solutions to implement experimental group 2-10, and specific experiment parameter is see such as following table 1:
Table 1, is separated from umbilical cord and cultivates component and content thereof contained by Digestive system and 0.25 trypsin-EDTA solutions the method for mescenchymal stem cell
Note:
1, in table 1, the number of DMEM substratum and F12 substratum only represents DMEM:F12=1:1.2, in table 1, the unit of collagenase is mg/ml, and the unit of Unidasa and Streptomycin sulphate is μ g/ml, and the unit of penicillin is 100u/ml, and the unit of 0.25% trypsin-EDTA solutions is ml.
In addition, also can adopt component and the ratio thereof of the Digestive system described in experimental group 1, in experimentation as umbilical cord tissue soak and digestion time, inoculating cell density, cleaning and centrifugal time be out of shape, concrete deformation experiment group is as follows:
Experimental group 11: umbilical cord clean for surface treatment is placed in dual anti-salt solution and soaks 18min, the multiplexing dual anti-saline soak 4 times of laying equal stress on, other experiment condition is identical with the experiment condition of experimental group 1.
Experimental group 12: umbilical cord clean for surface treatment is placed in dual anti-salt solution and soaks 15min, the multiplexing dual anti-saline soak 1 time of laying equal stress on, other experiment condition is identical with the experiment condition of experimental group 1.
Experimental group 13: the umbilical cord tissue shredded adds after PBS solution washs, and add Digestive system in experimental group 1, and digest at 37 ° of temperature, the time of digestion is 4h, and other experiment condition is identical with the experiment condition of experimental group 1.
Experimental group 14: the umbilical cord tissue shredded adds after PBS solution washs, and add Digestive system in experimental group 1, and digest at 37 ° of temperature, the time of digestion is 7h, and other experiment condition is identical with the experiment condition of experimental group 1.
Experimental group 15: the umbilical cord tissue shredded adds after PBS solution washs, and add Digestive system in experimental group 1, and digest at 37 ° of temperature, the time of digestion is 5h, and other experiment condition is identical with the experiment condition of experimental group 1.
Experimental group 16: the umbilical cord tissue shredded adds after PBS solution washs, and add Digestive system in experimental group 1, and digest at 37 ° of temperature, the time of digestion is 5.5h, and other experiment condition is identical with the experiment condition of experimental group 1.
Experimental group 17: the umbilical cord tissue shredded adds after PBS solution washs, and add Digestive system in experimental group 1, and digest at 37 ° of temperature, the time of digestion is 20h, and other experiment condition is identical with the experiment condition of experimental group 1.
Experimental group 18: the umbilical cord tissue shredded adds after PBS solution washs, and add Digestive system in experimental group 1, and digest at 37 ° of temperature, the time of digestion is 1 hour, and other experiment condition is identical with the experiment condition of experimental group 1.
Experimental group 19: need in primary cell culture process when cell grows to about 80% fusion, then carry out the operation of next step, other experiment condition is identical with the experiment condition of experimental group 1.
Experimental group 20: need in primary cell culture process when cell grows to about 60% fusion, then carry out the operation of next step, other experiment condition is identical with the experiment condition of experimental group 1.
Experimental group 21: need in primary cell culture process when cell grows to about 90% fusion, then carry out the operation of next step, other experiment condition is identical with the experiment condition of experimental group 1.
Experimental group 22: need in primary cell culture process when cell grows to about 75% fusion, then carry out the operation of next step, other experiment condition is identical with the experiment condition of experimental group 1.
Experimental group 23: need in primary cell culture process when cell grows to about 85% fusion, then carry out the operation of next step, other experiment condition is identical with the experiment condition of experimental group 1.
Controlled trial is carried out to the mescenchymal stem cell adopting the method separation and Culture from umbilical cord described in above-mentioned experimental group 1-23 to obtain or its preparation process and control group, cell growth metamorphosis, cell cultures surviving rate measure, and go forward side by side to carry out signature analysis to obtained mescenchymal stem cell.
Control group of the present invention is the mescenchymal stem cell adopting separation from Cord blood disclosed in patent CN 1630717A and cultivation mesenchymal stem/progenitor cells method separation and Culture to obtain.
Concrete detection method is as follows:
1, the mensuration of Growth of Cells metamorphosis
Experimental technique: above-mentioned experimental group 1-23 and control group are in the process of separation and Culture mescenchymal stem cell from umbilical cord and Cord blood, note observing substratum colour-change, and after acquisition primary cell, culture dish or the slide that is loaded with the required cell observed are seated in basis of microscopic observation cellular form and growing state thereof by every 24h, 48h and 72h.
Experimental result:
(1) cellular form in experimental group 1-23 and growing state as follows:
In experimental group 1-23, the process of Growth of Cells can be roughly: primary cell form is long shuttle-type, removes tissue block, distributes as seen under the microscope, differs in size and the cell colony of adherent growth after 6 days, and about 10 days cells reach more than 60% and merge;
After passage, adherent speed speeds, and has cell attachment as seen after 24h, and cellular form changes to fusiformis; After 48h, adherent amount of mononuclear cells increases, and cell is in the polygon with certain orientation; After 72h, have cell colony to be formed in attached cell as seen, in kytoplasm, particle is obvious, and along with cell fission propagation, the volume of colony becomes large, and its quantity also increases.Wherein experimental group 1 after Secondary Culture 72h, cell fission propagation is obviously.
Due to the difference of component proportion and experimental technique, have certain cell growth state and situation difference thereof between experimental group 1-23, concrete difference is as follows:
Between experimental group 1-5, cellular form and growing state thereof are without too big-difference, visible when collagenase consumption be 0.5-1.5mg/ml, the consumption of Unidasa is 4.5-5.5 μ g/ml, and collagenase and Unidasa floating of consumption in above-mentioned scope there is no too large impact for what be separated and cultivate the cellular form of mescenchymal stem cell and growing state from umbilical cord.
Experimental group 6-7 is due to the adjustment of DMEM substratum and F12 substratum ratio, and its vitro growth rates is slightly slower than the vitro growth rates of experimental group 1.
In experimental group 8-9, it is the adjustment of 0.25 trypsin-EDTA solutions consumption, comparatively speaking, the vitro growth rates of experimental group 9 is comparatively slower than experimental group 1, experimental group 8 and experimental group 10, and difference little between experimental group 1, vitro growth rates between 8 and 10, visible, when 0.25 trypsin-EDTA solutions consumption is 5-10ml, its vitro growth rates is optimum.
In experimental group 12, only adopt dual anti-saline soak 1 time, its vitro growth rates obtained is a little less than experimental group 1 and experimental group 11, and wherein experimental group 1 is also little with the difference of the vitro growth rates of experimental group 11.
Experimental group 13-17 is compared with experimental group 1, the change of its cellular form is little with speed of growth difference, and experimental group 18 is only 1h due to digestion time, the change of its cellular form is obviously worse than experimental group 1,13-16 with the speed of growth, and the cellular form between experimental group 1,13-16 changes with speed of growth difference also little, the digestion time of experimental group 17 is 20h, and digestion time is obviously more than the 4-6h of experimental group 13-16, and the quantity of its cultured cells has obvious minimizing.
Experimental group 1, experimental group 19 and cell in experimental group 22 be respectively grow to 70%, 80% and 75% merge after, carry out the operation of next step again, the speed of growth of its cell and form thereof are obviously better than experimental group 20, experimental group 21, experimental group 23 (cell grows to 60%, 90%, 85% respectively and merges), in experimental group 20,21 and 23, experimental group 23 cellular form and its speed of growth are obviously better than experimental group 20 and experimental group 21.That is, when cell reaches 70-80% fusion, the change of its cellular form is best with the speed of growth, and cytogamy degree becomes greatly or diminishes, and all can cause bad impact to its cellular form and its speed of growth.
(2) cellular form in control group and growing state as follows:
Primary cell form is fusiform, removes tissue block after 2 weeks, and after 3 weeks, cell can reach the fusion of more than 60%.After passage, 24h observes and has no obvious cell attachment, and cellular form change has no notable difference; After 48h, cell occurs adherent, but adherent scope is little; After 72h, cell occurs obviously adherent, and cell is the form growth of zonule fibrocyte sample colony shape, but in kytoplasm, particle is not obvious.
Interpretation of result: can find out from above-mentioned result and adopt method separation and Culture mescenchymal stem cell from umbilical cord described in experimental group 1-23 of the present invention, the change of its cellular form and speed thereof are better than control group.
2, the mensuration of cell cultures success ratio
Experimental technique: adopt trypan blue (Trypan Blue) to measure the cell of experimental group 1-23 and control group, during to evaluate its primary cultured cell, survival condition after cellular segregation, directly the cell of required detection is dyeed with 0.4% trypan blue, and examine under a microscope, to judge the viability of cell.
Experimental result:
Trypan Blue in experimental group 1-23 of the present invention is also not obvious, and in experimental group 1-23, the survival rate of institute's isolated cell can reach more than 99%.
The survival rate of control group institute isolated cell is 97-98%.
Interpretation of result: the survival rate in experimental group 1-23 of the present invention after cellular segregation is a little more than the cell survival rate of control group.
3, signature analysis is carried out to being separated from umbilical cord and cultivating mescenchymal stem cell
Experimental technique: in order to whether the cell being verified the umbilical cord source that the present invention obtains has the feature of mescenchymal stem cell, by the 3rd generation (P3) cell cultures of above-mentioned acquisition to during close to fusion, with containing 0.25% trypsinase-EDTA, cell dissociation is got off, PBS is adopted to wash 1 time, make single cell suspension, cell density is adjusted to l × l0
6/ ml.With PBS (phosphate buffered saline buffer) washed cell again, under room temperature, hatch 15min with monoclonal antibodies such as CD44, CD105, CD90, CD45, CD34.Fixing in 0.5ml is containing the PBS of 1% paraformaldehyde with re-suspended cell after PBS washed cell again.With isotype control monoclonal antibody determination context marker.Flow cytometer is analyzed.
Experimental result:
In experimental group 1-23, the cell obtained all shows the specific marker of strongly expressed mescenchymal stem cell as CD44, CD105, CD90 and CD73, and expresses negative to CD45, CD34, CD14, CD19 and HLA-DR.
The specific marker of the cell display strongly expressed obtained in control group comprises CD44, CD54, CD29, CD29e, and is shown as negative reaction to the antibody of CN64, CD106, CD51, CD61.
Interpretation of result: current internationally recognized MSCs phenotypic characteristic is: the antigen positive such as cell surface CD73, CD90, CD105, and the hematopoietic lineage developed by molecule such as CD14, CD19, CD34, CD45, HLA-DR are negative.Further, the MSCs of marrow, fatty tissue, umbilical cord tissue and Cord Blood-Derived is basically identical in the form in amplification procedure that goes down to posterity, the Flow cytometry amplifying cells that goes down to posterity is expressed CD29, CD44, CD105, CD166, CD146 positive mark and is occurred unimodal, and CD3, CD14, CD19, CD34, CD117, CD133, CD45, CD235a, HLA-DR all express feminine gender.
Visible, identical with the immunophenotype of mescenchymal stem cell from the immunophenotype of the cell in umbilical cord source in experimental group 1-23 of the present invention, this illustrates the feature by the cell obtained from umbilical cord separation and cultivation mescenchymal stem cell and to the method separation and Culture of chondrogenic differentiation described in the present invention with mescenchymal stem cell.
Further, the mescenchymal stem cell obtained in experimental group 1-23 of the present invention expresses CD44, and do not express hematopoietic cell and other cell surface mark, as CN34, also prove that the cell adopting method separation and Culture of the present invention is single mescenchymal stem cell.
embodiment 2: the differentiation of the mesenchymal stem cells into chondrocytes in umbilical cord source of the present invention
The differentiation of the mesenchymal stem cells into chondrocytes adopting the method described in experimental group 1 in embodiment 1 to obtain in the present embodiment
Particularly, the experimental group 1 of the present embodiment is as follows:
3rd generation (P3) cell is seeded in six well culture plates with certain cell density, the cover glass of preset sterilization in six orifice plates, is replaced by the Induction Transformation nutrient solution containing inducible factor after cultivating 24h, six orifice plates are placed in 5%CO
2, cultivate in saturated humidity 80%, 37 DEG C of incubators, change liquid 2-3 time as required weekly.
The component of this Induction Transformation nutrient solution comprises: containing DMEM/F12 nutrient solution and 10ng/ml transforming growth factor-beta I, 6.25 μ g/ml Regular Insulin, 100 μm of ol/ml dexamethasone, 1 μm of ol/ml Sodium.alpha.-ketopropionate and the 6.25 μ g/ml Transferrins,iron complexess of 5% foetal calf serum.
In fact experimental group 1 parameter can carry out the adjustment in certain limit, and concrete setting range is as follows:
The content range of dexamethasone can be 98-99 μm of ol/ml, 98.5-99.5 μm of ol/ml, 98-99.5 μm of ol/ml and 98.5-99 μm of ol/ml etc.
Also sodium β-glycerophosphate and vitamins D can be added further in the Induction Transformation nutrient solution of the present embodiment
3; Wherein, the content range of above-mentioned sodium β-glycerophosphate can be 0.01-1 μm of ol/ml, 0.01-0.5 μm of ol/ml, 0.01-1.5 μm of ol/ml, 0.01-2 μm of ol/ml, 0.5-2 μm of ol/ml and 1-2 μm of ol/ml etc.; Above-mentioned vitamins D
3content range can be 0.01-1 μm of ol/ml, 0.5-1.5 μm of ol/ml, 1-1.5 μm of ol/ml, 0.01-1.5 μm of ol/ml, 0.01-2 μm of ol/ml and 0.5-2 μm of ol/ml etc.
In other concrete experimental group of the present invention, preferred inoculating cell density is 2 × 10
5/ ml-1 × 10
6between/ml, also can more preferably 2 × 10
5/ ml-1 × 10
6any one interval or single numerical value between/ml, particularly, inoculum density also can be 7 × 10
4/ ml-1 × 10
7/ ml, all right 7 × 10
4/ ml-5 × 10
6/ ml, also can further with 7 × 10
4/ ml-1 × 10
6/ ml or 7 × 10
4/ ml-7 × 10
5/ ml or 7 × 10
4/ ml-5 × 10
5/ ml or 2 × 10
5/ ml-1 × 10
6/ ml or 5 × 10
5/ ml-5 × 10
6/ ml or 7 × 10
7/ ml-1 × 10
7the cell density scopes such as/ml are seeded in six well culture plates.
Above-mentioned mesenchymal stem cells into chondrocytes differentiation-inducing time is 20-24 days, and obtain chondrocyte, concrete number of days also can be 20-22 days or 21-23 days or 22-24 days or 22-23 days.
Except experimental group 1, applicant adopts the Induction Transformation nutrient solution of different parameters value to implement experimental group 2-19, and specific experiment parameter is see such as following table 2:
Wherein, from umbilical cord be separated and cultivate mesenchymal stem cells into chondrocytes differentiation Induction Transformation cultivate liquid ingredient and content as shown in table 2:
Table 2, the Induction Transformation being separated and cultivating mesenchymal stem cells into chondrocytes differentiation from umbilical cord cultivates liquid ingredient and content list thereof
Note:
1, the blending ratio that in table 2, DMEM/F12 nutrient solution is expressed as DMEM substratum and F12 substratum is 1:1.Containing 5% foetal calf serum in DMEM/F12 nutrient solution.
2, the unit of table 2 transforming growth factor beta I is ng/ml, and the unit of Regular Insulin and Transferrins,iron complexes is μ g/ml, dexamethasone, sodium β-glycerophosphate, Sodium.alpha.-ketopropionate and vitamins D
3unit be a μm ol/ml.
In addition, also can adopt experimental procedure and conditional request thereof in experimental group 1, be out of shape the inoculating cell density in experimentation, concrete deformation experiment group is as follows:
Experimental group 20: the 3 generation cell is with 1 × 10
6/ ml is seeded in six well culture plates, and other experiment condition is identical with the experiment condition of experimental group 1.
Experimental group 21: the 3 generation cell is with 8 × 10
5/ ml is seeded in six well culture plates, and other experiment condition is identical with the experiment condition of experimental group 1.
Experimental group 22: the 3 generation cell is with 5 × 10
6/ ml is seeded in six well culture plates, and other experiment condition is identical with the experiment condition of experimental group 1.
Experimental group 23: the 3 generation cell is with 1 × 10
7/ m l is seeded in six well culture plates, and other experiment condition is identical with the experiment condition of experimental group 1.
Experimental group 24: the 3 generation cell is with 2 × 10
5/ ml is seeded in six well culture plates, and other experiment condition is identical with the experiment condition of experimental group 1.
Experimental group 25: the 3 generation cell is with 7 × 10
4/ ml is seeded in six well culture plates, and other experiment condition is identical with the experiment condition of experimental group 1.
Experimental group 26: mesenchymal stem cells into chondrocytes differentiation-inducing time is 22 days, obtain chondrocyte, and carry out the frozen process of chondrocyte, other experiment condition is identical with the experiment condition of experimental group 1.
Experimental group 27: mesenchymal stem cells into chondrocytes differentiation-inducing time is 23 days, obtain chondrocyte, and carry out the frozen process of chondrocyte, other experiment condition is identical with the experiment condition of experimental group 1.
Experimental group 28: mesenchymal stem cells into chondrocytes differentiation-inducing time is 24 days, obtain chondrocyte, and carry out the frozen process of chondrocyte, other experiment condition is identical with the experiment condition of experimental group 1.
Experimental group 29: mesenchymal stem cells into chondrocytes differentiation-inducing time is 20 days, obtain chondrocyte, and carry out the frozen process of chondrocyte, other experiment condition is identical with the experiment condition of experimental group 1.
Experimental group 30: mesenchymal stem cells into chondrocytes differentiation-inducing time is 30 days, obtain chondrocyte, and carry out the frozen process of chondrocyte, other experiment condition is identical with the experiment condition of experimental group 1.
In above-mentioned experimental group the feature of each component and the mechanism of action specific as follows:
Transforming growth factor-beta is the multi-functional cyclin of a class, three kinds of forms are had in mammalian tissues, i.e. transforming growth factor-beta I, conversion growth factor beta II and transforming growth factor-beta III, wherein, transforming growth factor-beta I, β II and β III all have and participate in embry ogenesis and growth and regulate and control MSCs break up and rising in value, participate in the several functions such as wound healing, and the hyperplasia of energy significant stimulation chondrocyte, division, and differentiation.Transforming growth factor-beta I not only raises the expression (proteoglycan and II Collagen Type VI) of cartilage genes involved, also add the expression of endogenous transforming growth factor-beta I and corresponding acceptor.In addition, transforming growth factor-beta I also can increase
3 hthe integration of-thymus pyrimidine.In vitro, the effect of transforming growth factor-beta I couple of MSCs is the dependence mode with dosage, at 2-
10ng/mlthe formation of chondrocyte just effectively can be stimulated in scope.And short-term gives transforming growth factor-beta I MSCs just can be induced to break up under culture conditions, improve the quantity of chondrocyte.
As the present embodiment transforming growth factor beta I concentration and under the impact of other inducible factor that adds, the inducing effect of transforming growth factor-beta I strengthens to some extent, and is better than the differentiation-inducing effect of transforming growth factor-beta III.
In the process that cartilaginous precursor is formed, transforming growth factor-beta I can also induce original Derived from Mesenchymal Stem Cells to form chondrocyte, and can promote chondrocyte proliferation and maturation further, increases the effect of chondrocyte's synthesis and secretory protein polysaccharide.
Dexamethasone is the glucocorticosteroid of synthetic, can by strengthening the affinity of dexamethasone acceptor and genome target sequence, thus the expression of differentiation gene in recipient cell can be regulated and controled, thus improve the activity of alkaline phosphatase and the protein level of cartilage matrix tagged molecule, especially the level of II Collagen Type VI, dexamethasone is combined with transforming growth factor-beta I, the mescenchymal stem cell be separated from umbilical cord can be made to cartilage differentiation.
Regular Insulin can stimulate the propagation of chondrocyte's Colony forming and cell.
Containing 5% foetal calf serum and 10ng/ml transforming growth factor-beta I combined action also can inducing mesenchymal stem cell to Chondrocyte Differentiation.
Sodium β-glycerophosphate by alkaline phosphatase enzymic hydrolysis, can also induce the formation of bone mineral, and promotes the generation of lactic acid, strengthens the activity of alkaline phosphatase, strengthens the synthesis of protein and phosphatide.
Vitamins D
3a kind of open loop steroidal compounds, can antiproliferative effect in the cell cycle, thus bone forming and bone resorption reach the state of a balance.
In part Experiment group, although only add a small amount of sodium β-glycerophosphate and vitamins D
3, but the sodium β-glycerophosphate of the 0.5-1.5 added μm of ol/ml and 0.1-1 μm of ol/ml vitamins D
3with the synergy of 10ng/ml transforming growth factor-beta I, 98-99 μm ol/ml dexamethasone and 6.25 μ g/ml Regular Insulin, success inducing bone mesenchymal stem cell is to Chondrocyte Differentiation, compared to the Induction Transformation nutrient solution adding transforming growth factor-beta III, there is better changing effect.
To adopting in the present embodiment the method described in experimental group 1-30 be chondrocyte by mesenchyma stem cell differentiation induction, immunochemistry being carried out to chondrocyte and detects analysis.Concrete detection method is as follows:
1, aniline blue staining analysis
Experimental technique: take out the slide being loaded with and adopting the cell that method prepares described in experimental group 1-30 in embodiment 2, carry out PBS rinsing 2 times respectively, and adopt 4% paraformaldehyde room temperature to fix 20min, toluidine blue liquid effect 30min, washing, Glacial acetic acid liquid differentiation the several seconds, dehydration, transparent, resinene mounting.And with using chondrocyte as positive control
Experimental result is specifically as shown in table 3.
Interpretation of result:
As can be seen from above-mentioned result, the mesenchyma stem cell differentiation induction adopting the methods analyst in embodiment 2 described in experimental group 1-30 to cultivate to obtain secretes polysaccharide material for chondrocyte, can think that cell of the present invention fully can realize the function of chondrocyte fully.
2, immunohistochemical methods II Collagen Type VI qualification
Experimental technique: take out the slide being loaded with and adopting the cell that method prepares described in experimental group 1-30 in embodiment 2, PBS rinsing 2 times, 4% paraformaldehyde room temperature fixes 20min, Fresh 0.5%H
2o
2soaking at room temperature 30min is with deactivating endogenous peroxydase, drip confining liquid room temperature effect 20min and close nonspecific binding site, drip rabbit anti-human II Collagen Type VI antibody (primary antibodie) of 1:150, hatch 1-2h for 37 DEG C, drip biotinylated goat anti-rabbit IgG (two resist), hatch 20min for 37 DEG C, DAB develops the color 15min, Hematorylin is slightly redyed, dehydration, transparent, resinene mounting.Replace primary antibodie as blank, using chondrocyte as positive control with PBS.
Experimental result is specifically as shown in table 3.
Table 3, in the embodiment of the present invention 2, experimental group 1-30 immunochemistry detects analytical results list
Note: "○" represents and characterizes immunochemistry detected result, "×" represents and does not characterize immunochemistry detected result.
Interpretation of result:
As can be seen from above-mentioned result, adopt experimental group 1 in the embodiment of the present invention 2,5-22,24, methods analyst described in 26-29 cultivates the mesenchyma stem cell differentiation induction that obtains for chondrocyte and secretes II Collagen Type VI, also can think that cell of the present invention fully can realize the function of chondrocyte.Therefore in the present invention, the mescenchymal stem cell in umbilical cord source can differentiating cartilage-forming cell under proper condition.
Can find out in associative list 2 and table 3, characterize immunochemistry detected result in the embodiment of the present invention 2 in experimental group 2-4 and occur that abnormal reason is: the consumption of transforming growth factor-beta I is revised as 5ng/ml, 15ng/ml, 20ng/ml.Due to, the transforming growth factor-beta I of 10ng/ml provides suitable exogenous signals in the present embodiment 2, it can be combined with cellular endogenous sex factor, common inducing mesenchymal stem cell breaks up to chondrocyte direction, as transforming growth factor-beta I also can with dexamethasone combined action, induce the differentiation of whole mesenchymal stem cells into chondrocytes.When the consumption increase or less of transforming growth factor-beta I, the deleterious of its induction, is unfavorable for the conversion differentiation of mesenchymal stem cells into chondrocytes.
The reason that in the embodiment of the present invention 2, experimental group 23 and 25 characterizes the appearance of immunochemistry detected result abnormal is: the change in cell density of inoculation is excessive, due to cultured cartilage cell in vitro, under the inoculum density of different cells, can affect to Chondrocyte Differentiation, it even instead can break up or transforms to hypertrophic chondrocyte.During as cultured in monolayer in vitro under identical condition, because cell density is excessive or too small, the growth division of chondrocyte is through completely different with morphological function, and therefore, exception has appearred in immunochemistry detected result.
In the embodiment of the present invention 2, experimental group 30 characterizes immunochemistry detected result and also occurs exception, and its reason is: mesenchymal stem cells into chondrocytes differentiation-inducing time is 30 days, and differentiation-inducing overlong time, causes Chondrocyte Differentiation to occur abnormal.
In the embodiment of the present invention 2, mesenchyma stem cell differentiation induction can be all chondrocyte by experimental group 9-19, compared with experimental group 1 in the embodiment of the present invention 2, in experimental group 9-19, be better than experimental group 1 from the ability of cell proliferation of mesenchymal stem cells into chondrocytes differentiation.Adopt in the present invention
3h-TdR ((
3h-Thymidine, thymidine) mix cell analysis to react the multiplication capacity of chondrocyte.In the embodiment of the present invention 2, experimental group 9-19 is due to the sodium β-glycerophosphate of 0.5-1.5 μm of ol/ml that adds and 0.1-1 μm of ol/ml vitamins D
3with the synergy of 10ng/ml transforming growth factor-beta I, 98-99 μm ol/ml dexamethasone and 6.25 μ g/ml Regular Insulin, to mesenchyma stem cell differentiation induction being played for chondrocyte and promoting chondrocyte proliferation, thus reduce the level of cAMP in cell, make S/G
2the effect of the increase of M phase cell proportion.And in the experimental group 1 of the embodiment of the present invention 2, although can be also chondrocyte by mesenchyma stem cell differentiation induction, the multiplication capacity of its differentiation-inducing effect and chondrocyte be obviously worse than experimental group 9-19.Therefore, experimental group 9-19 has more excellent differentiation-inducing and cell proliferation effect relative to experimental group 1.
For experimental group 11, with difference in experimental group 1 of the present invention be, on the basis adding transforming growth factor-beta I and dexamethasone, in experimental group 11, add the sodium β-glycerophosphate of 2 μm of ol/ml, and the concentration of dexamethasone is adjusted to 98 μm of ol/ml.In experimental group 11, the sodium β-glycerophosphate of 2 μm of ol/ml and 10ng/ml transforming growth factor-beta I and 98 μm of ol/ml dexamethasone act synergistically and promote that mescenchymal stem cell is to the propagation of cartilage differentiation and chondrocyte.
For experimental group 14, be, on the basis adding transforming growth factor-beta I and dexamethasone, in experimental group 14, add the sodium β-glycerophosphate of 0.5 μm of ol/ml and the vitamins D of 0.5 μm of ol/ml with difference in experimental group 1 of the present invention
3, and the concentration of dexamethasone is adjusted to 99 μm of ol/ml.In experimental group 14, each component acts synergistically and promotes that mescenchymal stem cell is to the propagation of cartilage differentiation and chondrocyte.
embodiment 3: adopt prepared by the experimental group 12,14 in the embodiment of the present invention 2 and obtain
the mescenchymal stem cell treatment osteoarthritis obtained
By the method described in the experimental group 12,14 in the embodiment of the present invention 2 by the differentiation-inducing nutrient solution during the 3rd generation, cell added in embodiment 2 experimental group 12,14 of the mescenchymal stem cell adopting the method separation and Culture of experimental group 1 in the embodiment of the present invention 1 to obtain, form 1 × 10
8mescenchymal stem cell-nutrient solution the mixture of/ml.1ml cell suspending liquid is got (containing 1 × 10 with asepsis injector
8the mescenchymal stem cell of/ml), and directly transferred to the having in the hyaluronate sodium structure of certain three-dimensional configuration of freeze-drying, make it form uniform mixture.
(1) mild osteoarthritis patient: after toponarcosis, be injected directly in joint cavity with aseptic injection.
(2) moderate osteoarthritis patient: after toponarcosis, carries out cartilage injury debridement by arthroscope, uses the drill bit of 3-5mm to get out the hole of interval 2-3 centimetre, mixture is filled the hole on cartilaginous lesion site.
(3) severe osteoarthritic patients: after using narcotic, carries out cartilage injury debridement by synosteotomy, forms " bowl " shape groove, filled by mixture in the groove on cartilaginous lesion site at cartilage surface.
In the method using composition of the present invention treatment articular cartilage damage, the articular cartilage zones for the treatment of preferably by intraarticular peep operation spout row observe, damage field is processed into the state being convenient to perform the operation, then composition of the present invention is applied to damage field, confirms that whether it is firm preferably by arthroscope.Composition of the present invention can be mixed with the form being applicable to entering damage field in advance before being applied to damage field, or after it is applied to damage field, change over applicable state.
To be suspended in the state in suitable substratum, or with mixed with polymers after, above-mentioned cultured cells can be grafted directly to articular cartilage damage region, namely the method in the space formed by the not microbial film (such as periosteum) corresponding to damaged cartilage region to the cell infusion of mixed with polymers suspended in the medium can be used, close them to ooze out from the crack of periosteum to make the cell of suspension, the otch of suture operation.
Concrete treatment embodiment is as follows:
1, contrast before and after treatment
case 1: women, 65 years old, ICRS (International Cartilage Repair Society's cartilage injury
hierarchy system) mark II; Cartilage defect area 2.25cm
2
.
The culture condition described in experimental group 11 of the concrete employing embodiment of the present invention 2 carries out separation and Culture and forms 1 × 10
8mescenchymal stem cell-nutrient solution the mixture of/ml.
This Induction Transformation nutrient solution is made up of the sodium β-glycerophosphate containing the DMEM/F12 nutrient solution of 5% foetal calf serum and 10ng/ml transforming growth factor-beta I, 6.25 μ g/ml Regular Insulin, 98 μm of ol/ml dexamethasone, 1 μm of ol/ml Sodium.alpha.-ketopropionate, 6.25 μ g/ml Transferrins,iron complexess and 2 μm of ol/ml.
Case 1 treats nucleus magnetic resonance (MRI) situation in front joint as shown in Fig. 1 a, 2a.
Case 1 treats nucleus magnetic resonance (MRI) situation of posterior joint as shown in Fig. 1 b, 1e and Fig. 2 b-2e.
Contrast above-mentioned accompanying drawing can find out, it is more obvious that the chondrocyte adopting method described in the present invention to prepare in case 1 treats precartilage defect area, after the chondrocyte adopting the preparation method described in the present invention to prepare treats, cartilage defect region is obviously reduced.Particularly, Fig. 1 b-1e and Fig. 2 b-2e for respectively relative to after the joint treatment shown in Fig. 1 a and Fig. 2 a, every bimonthly to nucleus magnetic resonance (MRI) figure carrying out the joint obtained after identical operative site carries out identical inspection.
case 2: the male sex, 52 years old, ICRS marked II; Cartilage defect area 3.6cm
2
.
The culture condition described in experimental group 14 of the concrete employing embodiment of the present invention 2 carries out separation and Culture and forms 1 × 10
8mescenchymal stem cell-nutrient solution the mixture of/ml.
This Induction Transformation nutrient solution is by containing the sodium β-glycerophosphate of the DMEM/F12 nutrient solution of 5% foetal calf serum, 10ng/ml transforming growth factor-beta I, 6 μ g/ml Regular Insulin, 50 μm of ol/ml dexamethasone, 2 μm of ol/ml Sodium.alpha.-ketopropionates, 6 μ g/ml Transferrins,iron complexess, 0.5 μm of ol/ml and 0.5 μm of ol/ml vitamins D
3composition.
Case 2 treats nucleus magnetic resonance (MRI) situation in front joint as shown in Fig. 3 a, 4a.
Case 2 treats nucleus magnetic resonance (MRI) situation in front joint as shown in Fig. 3 b, 4b.
Contrast above-mentioned accompanying drawing can find out, the chondrocyte adopting method described in the present invention to prepare in case 2 before the treatment (as shown in Fig. 3 a and Fig. 4 a), subchondral bone capsule intracavity lesion is serious, cartilage defect region is larger, after the chondrocyte adopting the preparation method described in the present invention to prepare treats, as as shown in Fig. 3 b and Fig. 4 b, diseased region and cartilage defect region are obviously reduced.
case 3: women, 32 years old, ICRS marked II; Cartilage defect area 3.74cm
2
.
The culture condition described in experimental group 11 of the concrete employing embodiment of the present invention 2 carries out separation and Culture and forms 1 × 10
8mescenchymal stem cell-nutrient solution the mixture of/ml.
This Induction Transformation nutrient solution is made up of the sodium β-glycerophosphate containing the DMEM/F12 nutrient solution of 5% foetal calf serum, 10ng/ml transforming growth factor-beta I, 6.25 μ g/ml Regular Insulin, 2 μm of ol/ml Sodium.alpha.-ketopropionates, 6 μ g/ml Transferrins,iron complexess and 2 μm of ol/ml.
Case 3 treats nucleus magnetic resonance (MRI) situation in front joint as shown in Fig. 5 a, 6a.
Case 3 treats nucleus magnetic resonance (MRI) situation in front joint as shown in Fig. 5 b, 6b.
As shown in Fig. 5 a and Fig. 6 a, case 3 is before treating, cartilaginous areas has obvious defect part, after the chondrocyte adopting the preparation method described in the present invention to prepare treats, as as shown in Fig. 5 b and Fig. 6 b, treat protochondral defect part and substantially again form the cartilage with complete cambered surface.
case 4: the male sex, 57 years old, ICRS marked II; Cartilage defect area 6cm
2
.
The culture condition described in experimental group 14 of the concrete employing embodiment of the present invention 2 carries out separation and Culture and forms 1 × 10
8mescenchymal stem cell-nutrient solution the mixture of/ml.
This Induction Transformation nutrient solution is by containing the sodium β-glycerophosphate of the DMEM/F12 nutrient solution of 5% foetal calf serum, 10ng/ml transforming growth factor-beta I, 6.25 μ g/ml Regular Insulin, 100 μm of ol/ml dexamethasone, 1 μm of ol/ml Sodium.alpha.-ketopropionate, 6.25 μ g/ml Transferrins,iron complexess, 0.5 μm of ol/ml and 0.5 μm of ol/ml vitamins D
3composition.
Case 4 treats nucleus magnetic resonance (MRI) situation in front joint as shown in Fig. 7 a, 9a.
Case 4 treats nucleus magnetic resonance (MRI) situation in front joint as shown in Fig. 7 b, 9b.
As shown in Fig. 7 a, Fig. 8 a and Fig. 9 a, case 3 is before treating, and cartilage defects region is obvious, after the chondrocyte adopting the preparation method described in the present invention to prepare treats, as as shown in Fig. 7 b, Fig. 8 b and Fig. 9 b, treatment precartilage defect area part heals substantially.
As mentioned above, adopt the chondrocyte obtained from umbilical cord separation and cultivation mescenchymal stem cell and to the method for chondrogenic differentiation described in the present invention to be used for the treatment of osteoarthritis, there is good result for the treatment of.
Relative to prior art, first be separated from umbilical cord in the present invention and cultivate mescenchymal stem cell, and the mescenchymal stem cell of acquisition is carried out further Induction Transformation, may be used for the scale operation of the mescenchymal stem cell of so-called pluripotent cell, be significant in the treatment of the damaged tissue of regeneration difficulty.
Secondly, adopt the method described in the present invention, being separated from the umbilical cord of same donor and cultivating the survival rate be separated of mescenchymal stem cell to more than 99%, obtaining the pick-up rate of mescenchymal stem cell far above adopting separation and Culture in prior art.
Finally, adopt the preparation method described in the present invention, the specific marker being separated and cultivating mescenchymal stem cell strongly expressed from umbilical cord comprises CD44, CD105, CD90 and CD73, and CD45, CD34, CD14, CD19 and HLA-DR expresses feminine gender, the immunophenotype of the mescenchymal stem cell adopting method separation and Culture of the present invention to obtain is identical with the immunophenotype of mescenchymal stem cell, this illustrates the feature by the cell obtained from umbilical cord separation and cultivation mescenchymal stem cell and to the method separation and Culture of chondrogenic differentiation described in the present invention with mescenchymal stem cell.
To sum up, the chondrocyte adopting method of the present invention to prepare can be used in the treatment of human body cartilage defect, and can be beneficial to the scale operation of mescenchymal stem cell and chondrocyte, has larger application prospect.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within principle of the present invention, equivalent replacement and improvement etc. all should comprise within protection scope of the present invention.
Claims (9)
1. one kind to be separated from umbilical cord and to cultivate mescenchymal stem cell and to the method for chondrogenic differentiation, it comprises umbilical cord process, from umbilical cord and umbilical blood vessels isolated cell and carry out primary cell culture, passage cultivates and obtains mescenchymal stem cell and be chondrocyte by mesenchyma stem cell differentiation induction, it is characterized in that:
From umbilical cord and umbilical blood vessels isolated cell comprise adopt Digestive system umbilical cord and umbilical blood vessels are digested, this Digestive system is after DMEM substratum mixes with F12 substratum 1:1, then adds 0.5-1.5mg/ml collagenase, 4.5-5.5 μ g/ml Unidasa, 100u/ml penicillin and 100 μ g/ml Streptomycin sulphate mixed configuration and form;
Primary cell culture comprises employing cell culture fluid and cultivates, and this cell culture fluid is after DMEM substratum mixes with F12 substratum 1:1, then adds 100u/ml penicillin, 100 μ g/ml Streptomycin sulphates and 10% foetal calf serum;
Passage cultivate obtain mescenchymal stem cell be included in primary cell culture until cell grow to 70%-85% merge time, the cell culture fluid in the culture dish removing original cuiture drawn by dropper, after adopting PBS washing, 0.25% trypsin-EDTA solutions adding 5-10ml again digests, digestion also observation of cell at 37 DEG C, treat that cell body is tending towards becoming bowlder, the cell culture fluid adding equivalent immediately stops digestion; Cell is blown and beaten gently with suction pipe, until attached cell suspends, cell needed for centrifugal acquisition; Make single cell suspension with cell culture fluid, primary cell carries out Secondary Culture by 1:2 and obtains 1st generation passage cell; Make to use the same method repeat 2nd generation and the 3rd generation passage cell cultivation;
By mesenchyma stem cell differentiation induction for chondrocyte comprise by the 3rd generation cell with 7 × 10
4/ ml-1 × 10
7/ ml is seeded in culture plate, the cover glass of preset sterilization in culture plate, is replaced by the Induction Transformation nutrient solution containing inducible factor, culture plate is placed in 5%CO after cultivating 24h
2, cultivate in saturated humidity 80%, 37 DEG C of incubators, change liquid 2-3 time weekly;
Wherein, its this Induction Transformation nutrient solution comprises: containing the DMEM/F12 nutrient solution of 5% foetal calf serum, 10ng/ml transforming growth factor-beta I, 6-6.5 μ g/ml Regular Insulin, 98-100 μm ol/ml dexamethasone, 0.5-1.5 μm ol/ml Sodium.alpha.-ketopropionate and 6-6.5 μ g/ml Transferrins,iron complexes.
2. be separated from umbilical cord as described in claim 1 and cultivate mescenchymal stem cell and to the method for chondrogenic differentiation, it is characterized in that:
In umbilical cord treating processes, adopt dual anti-normal saline washing umbilical cord outside surface, this dual anti-salt solution is the physiological saline containing 100u/ml penicillin and 100 μ g/ml Streptomycin sulphates;
Umbilical cord treating processes comprises the sludged blood removing umbilical cord and umbilical blood vessels surface, and changes container, umbilical cord clean for surface treatment is placed in dual anti-salt solution and soaks 15-18min; Take out umbilical cord, change container, then repeat with dual anti-saline soak 2-4 time, each 15-18min; Change sterile chamber, umbilical cord segment, changes sterile chamber again, longitudinally cuts the amnion on umbilical cord surface along both sides open, longitudinally tears umbilical cord, after removing Umbilical artery and umbilical vein and other blood vessel extravasated blood, washs to salt water colorless limpid in dual anti-salt solution.
3. be separated from umbilical cord as described in claim 2 and cultivate mescenchymal stem cell and to the method for chondrogenic differentiation, it is characterized in that:
Isolated cell from umbilical cord and umbilical blood vessels also carries out primary cell culture and comprises and the umbilical cord and umbilical blood vessels that complete extravasated blood process being transferred to respectively in aseptic digestion vessel, is shredded by tissue by sterile scissors; Then the tissue shredded and PBS are mixed and centrifugal acquisition throw out, the Digestive system prepared is added in throw out, is transferred to after blowing afloat precipitation in another sterile petri dish, and put into 37 °, sterile culture case digestion 4-7h; By the solution digested and organize centrifugal rear acquisition throw out; Throw out cell culture fluid is resuspended, and be inoculated in 10cm culture dish, cell is now called primary cell, be placed in 37 DEG C, saturated humidity, containing 5%CO
2cultivate in incubator; 24h full dose changes liquid, and every 2-3 days change nutrient solution.
4. be separated from umbilical cord as described in claim 3 and cultivate mescenchymal stem cell and to the method for chondrogenic differentiation, it is characterized in that: this Digestive system is after DMEM substratum mixes with F12 substratum 1:1, then add 0.5-1mg/ml collagenase, 4.5-5 μ g/ml Unidasa, 100u/ml penicillin and 100 μ g/ml Streptomycin sulphate mixed configuration and form;
Passage cultivate obtain mescenchymal stem cell be also included in primary cell culture until cell grow to 70%-80% merge time, the cell culture fluid in the culture dish removing original cuiture drawn by dropper, after adopting PBS washing, then 0.25% trypsin-EDTA solutions adding 5ml digests;
And the 3rd generation cell with 2 × 10
5/ ml-1 × 10
6/ ml is seeded to the cultivation that the laggard mesenchymal stem cells in the ranks of culture plate is induced to differentiate into chondrocyte.
5. be separated from umbilical cord as described in claim 4 and cultivate mescenchymal stem cell and to the method for chondrogenic differentiation, it is characterized in that:
This Digestive system is after DMEM substratum mixes with F12 substratum 1:1, then adds 1mg/ml collagenase, 5 μ g/ml Unidasas, 100u/ml penicillin and 100 μ g/ml Streptomycin sulphate mixed configuration and form.
6. be separated from umbilical cord as described in claim 5 and cultivate mescenchymal stem cell and to the method for chondrogenic differentiation, it is characterized in that:
This Induction Transformation nutrient solution comprises: containing sodium β-glycerophosphate and the 0-1.5 μm of ol/ml vitamins D of the DMEM/F12 nutrient solution of 5% foetal calf serum and 10ng/ml transforming growth factor-beta I, 6-6.5 μ g/ml Regular Insulin, 98-100 μm ol/ml dexamethasone, 0.5-1.5 μm ol/ml Sodium.alpha.-ketopropionate, 6-6.5 μ g/ml Transferrins,iron complexes, 0-2 μm ol/ml
3.
7. be separated from umbilical cord as described in claim 6 and cultivate mescenchymal stem cell and to the method for chondrogenic differentiation, it is characterized in that:
This Induction Transformation nutrient solution comprises: containing sodium β-glycerophosphate and 0.5 μm of ol/ml vitamins D of the DMEM/F12 nutrient solution of 5% foetal calf serum and 10ng/ml transforming growth factor-beta I, 6.25 μ g/ml Regular Insulin, 98 μm of ol/ml dexamethasone, 1 μm of ol/ml Sodium.alpha.-ketopropionate, 6.25 μ g/ml Transferrins,iron complexess, 1.5 μm of ol/ml
3.
8. be separated from umbilical cord as described in claim 6 and cultivate mescenchymal stem cell and to the method for chondrogenic differentiation, it is characterized in that:
This Induction Transformation nutrient solution comprises: containing sodium β-glycerophosphate and 0.5 μm of ol/ml vitamins D of the DMEM/F12 nutrient solution of 5% foetal calf serum and 10ng/ml transforming growth factor-beta I, 6.25 μ g/ml Regular Insulin, 99 μm of ol/ml dexamethasone, 1 μm of ol/ml Sodium.alpha.-ketopropionate, 6.25 μ g/ml Transferrins,iron complexess, 0.5 μm of ol/ml
3.
9. be separated from umbilical cord as described in claim 6 and cultivate mescenchymal stem cell and to the method for chondrogenic differentiation, it is characterized in that:
This Induction Transformation nutrient solution comprises: containing the sodium β-glycerophosphate of the DMEM/F12 nutrient solution of 5% foetal calf serum and 10ng/ml transforming growth factor-beta I, 6.25 μ g/ml Regular Insulin, 98 μm of ol/ml dexamethasone, 1 μm of ol/ml Sodium.alpha.-ketopropionate, 6.25 μ g/ml Transferrins,iron complexess, 2 μm of ol/ml.
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| HK15112628.8A HK1212148A2 (en) | 2015-07-03 | 2015-12-23 | A method for the isolation and cultivation of mesenchymal stem cells from umbilical cords, and a method for the differentiation of the umbilical cord-derived mesenchymal stem cells into various mesenchymal tissues |
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| CN106635968A (en) * | 2016-10-14 | 2017-05-10 | 中卫华医（北京）生物科技有限公司 | Method of inducing and differentiating human umbilical cord derived mesenchymal stem cells into cartilage cells |
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| CN111690599A (en) * | 2020-07-17 | 2020-09-22 | 天晴干细胞股份有限公司 | Method for promoting mesenchymal stem cells to differentiate into chondroblasts |
| CN111826343A (en) * | 2020-07-23 | 2020-10-27 | 北京中卫医正科技有限公司 | Cell culture solution for enhancing induced cartilage differentiation, method and application |
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