CN103361308B - A kind of method of mass-producing amplification mesenchymal stem cells MSCs - Google Patents
A kind of method of mass-producing amplification mesenchymal stem cells MSCs Download PDFInfo
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Abstract
The present invention relates to the method for a kind of mass-producing amplification mesenchymal stem cells MSCs.Disclose a kind of method utilizing bio-reactor microcarrier low serum amplification mesenchymal stem cells MSCs of optimization, described method can turn out a large amount of mesenchymal stem cells MSCs efficiently, and the mesenchymal stem cells MSCs vigor obtained is good.Method of the present invention is applicable to large scale fermentation and produces, and can obtain a large amount of mesenchymal stem cells MSCs, for the application such as experimental study, organizational project in the short period of time.
Description
Technical field
The invention belongs to biological technical field; More specifically; The present invention relates to a kind of method that mesenchymal stem cells MSCs is prepared in mass-producing.
Background technology
Be present in the mesenchymal stem cells MSCs (bonemarrowMesenchymalStemCells in marrow, bmMSCs) be a kind of adult stem cell, there is multi-lineage potential, scleroblast, adipocyte, chondrocyte and neurocyte etc. can be induced to differentiate into.Due to autotransplantation can be carried out, therefore become study hotspot in recent years.BmMSCs draws materials conveniently, is easy to amplification in vitro and the many differentiation potentials of long-term maintenance, can carries out autotransplantation and there is not tissue matching and immunological rejection, be considered to seed cell best in organizational project.
Mesenchymal stem cells MSCs needs a large amount of cells for clinical, organizational project, but the stem cell population obtained from marrow is limited, can not be satisfied with the cell concentration needed for organizational project.Because mesenchymal stem cells MSCs needs adherent growth, laboratory uses the culturing bottle not only consumptive material consumption that increases large, and the time pay also many.Bio-reactor is generally used for extensive amplifying cells, can a large amount of cell of disposable acquisition, has both saved a large amount of consumptive materials, and has in turn saved a lot of time.But this area does not also have successfully extensive amplification to obtain the precedent of mesenchymal stem cells MSCs at present.
Therefore, this area in the urgent need to studying the extensive amplification method of mesenchymal stem cells MSCs, to meet needed for scientific research and clinical application.
Summary of the invention
The object of the present invention is to provide the method for a kind of mass-producing amplification mesenchymal stem cells MSCs.
In a first aspect of the present invention, provide a kind of method of cultivating mesenchymal stem cells MSCs, described method comprises:
(1) mesenchymal stem cells MSCs is carried out low serum domestication, obtain the mesenchymal stem cells MSCs of low serum domestication;
(2) mesenchymal stem cells MSCs of low serum domestication step (1) obtained is with 2.3-2.7 × 10
5cell/ml (preferably 2.4-2.6 × 10
5cell/ml; More preferably 2.5 × 10
5cell/ml) cell density be incubated in the substratum of serum-concentration 0.8-4% (v/v) (preferably 1-3% (v/v)), also adding density in described substratum is that 3.5-4.5mg/ml is (more preferably for 3.8-4.2mg/ml; Be more preferably 4mg/ml) microcarrier cytodex3; Cultivate 4-10 days;
(3) acquisition mesenchymal stem cells MSCs is separated.
In a preference, in step (1), the method for described low serum domestication is: joined successively by mesenchymal stem cells MSCs in the substratum that serum content successively decreases successively, make mesenchymal stem cells MSCs adapt to low serum environment.
In another preference, described low serum acclimation method is:
(a) by cell with 1 × 10
5cell/ml density is seeded in the substratum of 10% (v/v) serum, cultivates 48h, gathers in the crops the cell converged close to 90%;
B () cell is again with 2 × 10
5cell/ml density is seeded in the substratum of 7% (v/v) serum, when Growth of Cells is to when converging close to 90%, and trysinization harvested cell, and be seeded to identical density in the substratum of 7% serum and cultivate;
C the cell of () etc. (b) regrows to close to when converging, the flow process of the cell of results repetition (b) be seeded to successively in the substratum of 5% (v/v), 3% (v/v), 1% (v/v) serum and cultivate.
In another preference, in step (2), in 20-28 hour (as 24 hours) of initial cultivation, serum content 3 ± 0.5% (v/v) in substratum; Wild Oryza species in serum content 1 ± 0.2% (v/v) (more preferably 1 ± 0.1% (v/v)).
In another preference, change substratum 50% every 0.5-2 days (as 1 day).
In another preference, in step (2), before initial cultivation 10-14 hour (as 11-13 hour), with half volume culture medium culturing, also namely in this stage, in substratum, cell and microcarrier cytodex3 are in the density of multiplication, if cell is 4.6-5.4 × 10
5cell/ml; Or microcarrier cytodex3 is 7-9mg/ml; After initial cultivation 10-14 hour, with full volumetric culture medium culturing, also namely in this stage, cell is 2.3-2.7 × 10
5cell/ml; Or microcarrier cytodex3 is 3.5-4.5mg/ml.
In another preference, in step (2), within first 12 hours, carry out intermittent stirring, 3min/h; Within 13-48 hour, cultivate with 35rpm rotating speed; Within 49-96 hour, cultivate with 45rpm rotating speed; 97 hours and cultivate with 50rpm rotating speed afterwards; Or
In step (2), controlling dissolved oxygen (DO) is 40 ± 10% (v/v); Or
In step (2), control ph is respectively and 7.2 ± 0.2.
In another preference, described substratum is DMEM/F12 substratum.
In another preference, mesenchymal stem cells MSCs is cultivated in the system of 10mL-100L (as 100mL, 1.5L, 5L, 10L, 50L).
In another preference, in step (3), being separated the method obtaining mesenchymal stem cells MSCs is: from substratum, be separated the microcarrier cytodex3 having attached cell, washing, with tryptic digestion, interval concussion, to make cell come off from microcarrier cytodex3, is separated and removes microcarrier cytodex3.
In another aspect of this invention, provide a kind of method being prepared into chondrocyte, described method comprises:
I () basis above arbitrary described method obtains mesenchymal stem cells MSCs;
(ii) mesenchymal stem cells MSCs of (i) is carried out chondroblast differentiation-inducing, thus obtain chondroblast.
In a preference, cultivate with the inducing culture containing dexamethasone and TGF-β 3; More preferably, ancillary component proline(Pro), xitix, Sodium.alpha.-ketopropionate, ITS is also comprised in the inducing culture of described chondroblast.More preferably, the inducing culture of described chondroblast is DMEM/F12+10ng/mlTGF-β 3+0.1 μM of dexamethasone+0.35mM proline(Pro)+50 μ g/ml xitix+1mM Sodium.alpha.-ketopropionate+1% (v/v) ITS.
In another aspect of this invention, provide one to prepare osteoblastic method, described method comprises:
I () basis above arbitrary described method obtains mesenchymal stem cells MSCs;
(ii) mesenchymal stem cells MSCs of (i) is carried out scleroblast differentiation-inducing, thus obtain scleroblast.
In a preference, cultivate with the osteoblast induction medium containing dexamethasone and sodium β-glycerophosphate; More preferably, ancillary component xitix, ITS is also comprised in described osteoblastic inducing culture.More preferably, the inducing culture of described chondroblast is DMEM+0.1 μM of dexamethasone+10mM sodium β-glycerophosphate+50 μ g/ml xitix+1% (v/v) ITS.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
The mesenchymal stem cells MSCs (14 days) that Fig. 1, separation and purification obtain.
The mesenchymal stem cells MSCs of Fig. 2, low serum domestication.
Fig. 3, bio-reactor increase the mesenchymal stem cells MSCs obtained.
The immunophenotypic characterization of Fig. 4, mesenchymal stem cells MSCs.
The qualification of Fig. 5, the differentiation of mesenchymal stem cells MSCs chondroblast, A group is experimental group, and B group is control group.Wherein,
1. Stigma Croci-O-BG dyeing;
2. Toluidine blue staining;
3. II Collagen Type VI immunocytochemical stain.
The qualification of Fig. 6, mesenchymal stem cells MSCs osteoblast differentiation, A group is experimental group, and B group is control group.Wherein,
1. alkaline phosphatase (ALP) dyeing;
2. sodium alizarinsulfonate Mineral nodules dyeing;
3. NTx immunocytochemical stain.
Embodiment
The present inventor is through deep research, disclose a kind of method utilizing bio-reactor microcarrier low serum amplification mesenchymal stem cells MSCs of optimization, described method can turn out a large amount of mesenchymal stem cells MSCs efficiently, and the mesenchymal stem cells MSCs vigor obtained is good.Method of the present invention is applicable to large scale fermentation and produces, and can obtain a large amount of mesenchymal stem cells MSCs, for the application such as experimental study, organizational project in the short period of time.
Cultivate mesenchymal stem cells MSCs
Present invention is disclosed a kind of method utilizing bio-reactor microcarrier low serum amplification mesenchymal stem cells MSCs of optimization, comprise: mesenchymal stem cells MSCs is carried out low serum domestication by (1), obtain the mesenchymal stem cells MSCs of low serum domestication; (2) mesenchymal stem cells MSCs of low serum domestication step (1) obtained is with 2.3-2.7 × 10
5the cell density of cell/ml is incubated in the substratum of serum-concentration 0.8-4% (v/v), also adds the microcarrier cytodex3 that density is 3.5-4.5mg/ml in described substratum; Cultivate 4-10 days; (3) acquisition bone marrow-drived mesenchymal stem is separated.
In current prior art, because it is to the preference of serum free culture system, the cultivation of mesenchymal stem cells MSCs still depends on the serum (usual 10% (v/v)) adding higher concentration in the medium.But, in time carrying out large scale culturing, the serum content of high density like this certainly will increase the cost producing mesenchymal stem cells MSCs greatly, and therefore this requirement of high density serum greatly limit the development process of mesenchymal stem cells MSCs large scale culturing technology.In view of above-mentioned factor, present inventor has performed deep research, adopt mesenchymal stem cells MSCs low serum acclimatization technology to overcome its dependency to serum.The method of described low serum domestication is: joined successively by mesenchymal stem cells MSCs in the substratum that serum content successively decreases successively, make mesenchymal stem cells MSCs adapt to low serum environment.
As optimal way of the present invention, described low serum acclimation method is: (a) by cell with 1 × 10
5cell/ml density is seeded in the substratum of 10% (v/v) serum, cultivates 48h, gathers in the crops the cell converged close to 90%; B () cell is again with 2 × 10
5cell/ml density is seeded in the substratum of 7% (v/v) serum, when Growth of Cells is to close to when converging, and trysinization harvested cell, and be seeded to identical density in the substratum of 7% serum and cultivate; C the cell of () etc. (b) regrows to close to when converging, the flow process of the cell of results repetition (b) be seeded to successively in the substratum of 5% (v/v), 3% (v/v), 1% (v/v) serum and cultivate.By this acclimation method, can obtain and at the mesenchymal stem cells MSCs of low serum ambient growth, thus can greatly save the cost of large scale culturing.
As used herein, described " converging " refers in monolayer cell culture, the state that the edge of all cells all contacts with other cells.Described " converging close to 90% " represents the degree of converging of cell, is that phalangeal cell is bred and formed 80-90% degree of converging, the preferably monolayer cell of 85-90% degree of converging in culture.Cell confluency degree determines it is well known to those skilled in the art.
The present inventor finds under study for action, mesenchymal stem cells MSCs is when utilizing microcarrier to carry out attaching cultivation, cell density and microcarrier consumption are the influence factors of outbalance, and too low or too high cell density all significantly can hinder the Growth and reproduction of mesenchymal stem cells MSCs.Therefore, the present inventor, through repetition test, optimizes cell density and microcarrier consumption.The mesenchymal stem cells MSCs of being tamed by low serum is with 2.3-2.7 × 10
5the cell density of cell/ml is incubated at serum-concentration 0.8-4% (v/v) and containing in the substratum of 3.5-4.5mg/ml microcarrier cytodex3, this cultural method can make mesenchymal stem cells MSCs breed with good state growth.
As optimal way of the present invention, after low serum domestication, when carrying out large scale culturing, in 20-28 hour of initial cultivation, serum content 3 ± 0.5% (v/v) in substratum; Wild Oryza species in serum content 1 ± 0.2% (v/v) (more preferably 1 ± 0.1% (v/v)).This is conducive to rapid attachment and the growth of cell.
As optimal way of the present invention, after low serum domestication, when carrying out large scale culturing, changed substratum 50% every 0.5-2 days.Because the cell attachment of the overwhelming majority is in microcarrier cytodex3, therefore, the liquid that changes of substratum is comparatively easy to carry out.The liquid that changes of substratum is conducive to cell and obtains fresh substratum, for cell provides more sufficient and good nutritive ingredient, is conducive to the growth of cell.
As optimal way of the present invention, after low serum domestication, when carrying out large scale culturing, 10-14 hour before initial cultivation, with half volume culture medium culturing, also namely in this stage, in substratum, cell and microcarrier cytodex3 are in the density of multiplication, if cell is 4.6-5.4 × 10
5cell/ml; Or microcarrier cytodex3 is 7-9mg/ml; After initial cultivation 10-14 hour, with full volumetric culture medium culturing, also namely in this stage, cell is 2.3-2.7 × 10
5cell/ml; Or microcarrier cytodex3 is 3.5-4.5mg/ml.First take the half volume culture medium culturing of for some time, this section of time inner cell density can be made to increase, be conducive to the attaching of cell.And full volumetric cultivation is carried out afterwards, cell better can be contacted with medium component, be conducive to the growth of cell and copy.
The present inventor also optimizes the other factors in culturing process, and such as, rotating speed for bio-reactor is optimized, and adopts following condition: within first 12 hours, carry out intermittent stirring, 3min/h; Within 13-48 hour, cultivate with 35rpm rotating speed; Within 49-96 hour, cultivate with 45rpm rotating speed; 97 hours and cultivate with 50rpm rotating speed afterwards.
Adopt method of the present invention, achieve the scale operation of mesenchymal stem cells MSCs.Described mesenchymal stem cells MSCs can be cultivated in the system of 10mL-100L (as 100mL, 1.5L, 5L, 10L, 50L).
The substratum of the mesenchymal stem cells MSCs that method of the present invention adopts, except the restriction of foregoing serum-concentration, other nutritive ingredient with reference to mesenchymal stem cells MSCs substratum disclosed in prior art, can be not limited to DMEM substratum.Preferably, described substratum is DMEM/F12 substratum.
From substratum, the method for separating bone marrow mesenchymal stem has no particular limits.Cultivate because the present invention adopts microcarrier to attach, therefore first can isolate the microcarrier having attached cell from substratum, then from isolated cell microcarrier, as the mode isolated cell by digestion (digest).A kind of optimal way is: from substratum, be separated the microcarrier cytodex3 having attached cell, washing, and with tryptic digestion, interval concussion, to make cell come off from microcarrier cytodex3, is separated and removes microcarrier cytodex3.
Mesenchymal stem cells MSCs after amplification can be digested frozen after collecting or directly apply.
Method of the present invention, utilize bio-reactor microcarrier culturing cell, for cell provides the growing environment of a 3 D stereo, effectively can promote the propagation of cell, the amplification efficiency of cell is high, and can obtain at short notice in a large number can for the mesenchymal stem cells MSCs of application.
Be prepared into chondrocyte or scleroblast
The mesenchymal stem cells MSCs that method of the present invention obtains, has good vigor, can be induced to differentiate into chondrocyte or scleroblast further.
For from the condition of mesenchymal stem cells differentiation needed for chondrocyte and composition, those skilled in the art know.Therefore, utilizing after method of the present invention obtains mesenchymal stem cells MSCs, technology well known to those skilled in the art can be adopted induce and make it to form chondrocyte.Wherein, dexamethasone promotes that mesenchymal stem cells MSCs is to the neccessary composition of bone cell differentiation; TGF-β 3 promotes that mesenchymal stem cells MSCs is to the neccessary composition of Chondrocyte Differentiation; Some cofactors such as proline(Pro), xitix, Sodium.alpha.-ketopropionate can provide environment preferably for mesenchymal stem cells MSCs to Chondrocyte Differentiation; And ITS (full name Regular Insulin, Transferrins,iron complexes and Sodium Selenite mixture) is a class medium additives, be conducive to the growth of cell.As an example of the present invention, the inducing culture of described chondroblast is DMEM/F12+10ng/mlTGF-β 3+0.1 μM of dexamethasone+0.35mM proline(Pro)+50 μ g/ml xitix+1mM Sodium.alpha.-ketopropionate+1% (v/v) ITS.
For from the condition of mesenchymal stem cells differentiation needed for scleroblast and composition, those skilled in the art know.Therefore, utilizing after method of the present invention obtains mesenchymal stem cells MSCs, technology well known to those skilled in the art can be adopted induce and make it to be formed into osteocyte.Wherein, dexamethasone promotes that mesenchymal stem cells MSCs is to the neccessary composition of bone cell differentiation; Sodium β-glycerophosphate is the neccessary composition promoting bone mesenchymal stem cell to osteoblast differentiation; Some cofactors such as xitix can provide environment preferably for bone mesenchymal stem cell to osteoblast differentiation; And ITS (full name Regular Insulin, Transferrins,iron complexes and Sodium Selenite mixture) is medium additives, promote that cell is to the picked-up of nutritive substance and the growth of self.As an example of the present invention, the inducing culture of described chondroblast is DMEM+0.1 μM of dexamethasone+10mM sodium β-glycerophosphate+50 μ g/ml xitix+1% (v/v) ITS.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, conveniently condition such as J. Pehanorm Brooker etc. is write usually, Molecular Cloning: A Laboratory guide, Science Press, the condition described in 2002, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.
Embodiment 1, mesenchymal stem cells MSCs separation and Culture, low serum are tamed
1, mesenchymal stem cells MSCs separation and Culture
The marrow of mouse tibia and femur is gathered under aseptic condition, after crossing 200 order stainless steel mesh screens, volume ratio by 1: 1 is added on lymphocyte separation medium (1.073g/mL), upper strata is with the centrifugal 30min of 3000r/min, collect the mononuclearcell of cloud tunica albuginea layer in another centrifuge tube, DMEM/F12 washs 2 times, and the centrifugal 5min of 1000r/min, abandons supernatant liquor.With the DMEM/F12 re-suspended cell containing volume fraction being 10% foetal calf serum (unless otherwise indicated, the percentage ratio of serum-concentration by volume), with 1 × 10
5/ cm
2(being square centimeter) inoculating cell, in culturing bottle, is placed in 37 DEG C, volume fraction is the CO of 5%
2in saturated humidity constant incubator, and be labeled as primary (P
0).Every 3 days (d) changes liquid 1 time later, and the adherent fusion of primary cell in about 14 days reaches about 90%, is arranged with obvious directivity, presents typical vortex shape, as Fig. 1.After trysinization, in 1: 3 ratio sub-bottle Secondary Culture (substratum is still the DMEM/F12 of 10% foetal calf serum), and be labeled as P1 for cell.In Secondary Culture process, cell reaches had digestive transfer culture after 90% fusion, and is labeled as P2 generation, by that analogy.
2, the low serum domestication of mesenchymal stem cells MSCs
Get the bmMSCs being passaged to the third generation (P3) and converging close to 90%, PBS liquid rinses 1 time, and with the tryptic digestion 2-3min of 0.25%, 800rpm is centrifugal, and 5min abandons supernatant liquor, and resuspended rear counting is with 1 × 10
5cell/ml density is seeded in the substratum containing serum and cultivates, and in substratum, serum-concentration successively decreases by 10% to 1%, carrys out low serum domestication cell with this.Concrete experiment flow is:
(1) by cell with 1 × 10
5cell/ml density is seeded in the DMEM/F12 substratum of 10% serum, and the tryptic digestion cultivating 48h, 0.25% (w/v) gathers in the crops the cell converged close to 90%.
(2) cell is again with 2 × 10
5cell/ml density is seeded in the DMEM/F12 substratum of 7% serum, when Growth of Cells is to when converging close to 90%, and 0.25% (w/v) trysinization harvested cell, and be seeded to identical density in the substratum of 7% serum and repeat cultivation one time.
Etc. (3) cell regrows to when converging close to 90%, and the flow process that the cell gathered in the crops by the tryptic digestion of 0.25% (w/v) repeats (2) is seeded in the DMEM/F12 substratum of 5%, 3%, 1% serum successively cultivates.
When the serum-concentration in substratum is down to 1%, cell still can keep good growth vigor, as Fig. 2.By frozen for the cell harvesting of 1% serum DMEM/F12 culture medium culturing, as seed cell.
Embodiment 2, bio-reactor amplification mesenchymal stem cells MSCs
1,100ml rolling bottle optimizes optimal culture condition
Collect the cell of the low serum domestication of aforementioned acquisition, with 1.5 × 10
5cell/ml, 2.0 × 10
5cell/ml, 2.5 × 10
5cell/ml and 3.0 × 10
5the multiple different densities such as cell/ml are inoculated in rolling bottle, microcarrier cytodex3 density be the multiple different interpolation density such as 3mg/ml, 4mg/ml and 5mg/ml to carry out integrated survey, a part of cell and microcarrier concrete add combine as shown in table 1.In rolling bottle, DMEM/F12 substratum working volume is 80ml.Initial culture conditions is that microcarrier is hatched in advance and spent the night in rolling bottle, DMEM/F12 substratum containing 3% (v/v) FBS in rolling bottle, cell was seeded in half volume DMEM/F12 substratum (40ml) and carried out intermittent stirring 12h next day, i.e. 3min/h (stir, then rest 57 minutes for first 3 minutes per hour).Add DMEM/F12 substratum after 12h to full volumetric (80ml), adjustment of rotational speed is that after 35rpm, 48h, rotating speed is adjusted to 45rpm, is adjusted to 50rpm again after 96h.In whole culturing process, in DMEM/F12 substratum, serum-concentration maintains 1% (v/v), changes liquid every 24h half volume.
The inoculum density of table 1, cell and microcarrier combines
By the optimization to cell-seeding-density and microcarrier density, when cell is with 2.5 × 10
5when the inoculation of cell/ml density and microcarrier density are 4mg/ml, the final density of cell can reach 2.06 × 10
6cell/ml.
2, stirring reactor mass-producing amplification mesenchymal stem cells MSCs
Culture condition in stirring reactor (1.5L) adopts the optimum culture condition of rolling bottle, and the working volume of reactor is 600ml.First will collect the cell of the low serum domestication of aforementioned acquisition, PBS liquid rinses 1 time, and with the tryptic digestion 2-3min of 0.25%, 800rpm is centrifugal, and 5min abandons supernatant liquor, and resuspended rear counting is with 2.5 × 10
5cell/ml density is seeded in the reactor containing DMEM/F12 substratum and cultivates, and microcarrier density is 4mg/ml; Further, intermittent stirring is carried out, i.e. 3min/h in front 12h half volume medium (300ml).Substratum is added into full volumetric (600ml) after 12h, adjustment of rotational speed is that after 35rpm, 48h, rotating speed is adjusted to 45rpm, is adjusted to 50rpm again after 96h.Note: in this section, 2.5 × 10
5the calculating of the cell density of cell/ml and the microcarrier density of 4mg/ml calculates with full volumetric substratum; Also namely when half volume is cultivated, actual cell density 5 × 10
5cell/ml; Actual microcarrier density 8mg/ml; Half volume of appropriate time is cultivated and is conducive to cell attachment in microcarrier.
In whole culturing process, in substratum, before serum-concentration, 24h maintains 3% (v/v) (calculating with half volume medium), control after 24h, 1% (v/v) (calculating with full volumetric substratum), to change liquid 50% every 24h.Because rolling bottle does not possess the function of on-line monitoring culture condition, and stirring type bioreactor can pass through four gas (CO in whole culturing process
2, N
2, O
2with air) auto-control, control dissolved oxygen (DO) with pH value be respectively 40% (v/v) and 7.2, thus maintenance cell cultures optimum environment, therefore, can 2.6 × 10 be reached by the density of the culturing cells of about 5 days
6cell/ml.In whole culturing process, the cell state in each stage is as Fig. 3.
The qualification of embodiment 3, mesenchymal stem cells MSCs, comprises values of immunophenotyping and differentiation capability is identified
1, Flow cytometry Immunophenotyping
Get and attached the microcarrier of cell when cell density is higher in reactor, PBS liquid washes 2 times, with the tryptic digestion 2-3min of 0.25% (w/v), and interval concussion is to make cell come off from microcarrier, the PBS adding 3% (v/v) serum stops digestion, the static microcarrier that allows precipitates, and draws the supernatant liquor containing cell, each EP pipe 5 × 10 of packing after counting
5individual cell, add anti-CD29-PE monoclonal antibody (purchased from BD), CD90-PE monoclonal antibody (purchased from BD) and CD34-PE monoclonal antibody (purchased from BD) respectively, hatch 30min for 4 DEG C, wash 1-2 time with PBS (pH7.2-7.4), add damping fluid resuspended, upper machine testing.
Shown in qualification result, the Flow cytometry result of bmMSCs surface antigen CD29, CD90 is positive, and the detected result of surface antigen CD34 is negative, and detected result shows that the cell that microcarrier increases still keeps good physiologically active, as Fig. 4.
2, qualification after the one-tenth cartilage of cell, Osteoblast Differentiation potential and differentiation
Get and attached the microcarrier of cell when cell density is the highest in reactor, PBS liquid washes 2 times, with the tryptic digestion 2-3min of 0.25% (w/v), and interval concussion is to make cell come off from microcarrier, the PBS added containing 3% (v/v) serum stops digestion, and the static microcarrier that allows precipitates, draw the supernatant liquor containing cell, centrifugally remove supernatant, add following chondroblast or osteoblast induction medium, regulate cell concn to 1 × 10
6cell/ml, is inoculated in 6 orifice plates and cultivates 15 days.
(1) chondrocyte induction differentiation, is become
Chondrocyte's inducing culture is: DMEM/F12+10ng/mlTGF-β 3+0.1 μM of dexamethasone+0.35mM proline(Pro)+50 μ g/ml xitix+1mM Sodium.alpha.-ketopropionate+1% (v/v) ITS, set up control group simultaneously, be namely inoculated in the DMEM/F12 substratum containing 10% serum with identical cell density.After inducing culture 15 days, carry out Toluidine blue staining, kind premium-O-BG dyeing and II Collagen Type VI immunocytochemical stain, basis of microscopic observation.Compared with control group (B), experimental group (A) coloration result all in strong positive, as Fig. 5.
The above results illustrates, the mesenchymal stem cells MSCs that the present invention cultivates acquisition successfully can be induced to differentiate into chondroblast.
(2), Osteoinductive differentiation
Osteoblast induction medium is: DMEM+0.1 μM of dexamethasone+10mM sodium β-glycerophosphate+50 μ g/ml xitix+1%ITS.Set up control group simultaneously, be namely inoculated in the DMEM/F12 substratum containing 10% serum with identical cell density.After inducing culture 15 days, carry out the dyeing of sodium alizarinsulfonate Mineral nodules, alkaline phosphatase staining and II Collagen Type VI immunocytochemical stain, microscopy.Visible stain all in strong positive, as Fig. 6.
The above results illustrates, the mesenchymal stem cells MSCs that the present invention cultivates acquisition successfully can be induced to differentiate into scleroblast.
In sum, mesenchymal stem cells MSCs prepared by aforesaid method has stable physiologically active, may be used in follow-up experimental study or other Application Areass.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (9)
1. utilize stirring type bioreactor to cultivate a method for mesenchymal stem cells MSCs, it is characterized in that, described bio-reactor is 100mL-5L bio-reactor, and described method comprises:
(1) mesenchymal stem cells MSCs is carried out low serum domestication, obtain the mesenchymal stem cells MSCs of low serum domestication;
(2) mesenchymal stem cells MSCs of low serum domestication step (1) obtained is with 2.3-2.7 × 10
5the cell density of cell/ml is incubated in the substratum of serum-concentration 0.8-4% (v/v), also adds the microcarrier cytodex3 that density is 3.5-4.5mg/ml in described substratum; Cultivate 4-10 days;
In 20-28 hour of initial cultivation, serum content 3 ± 0.5% (v/v) in substratum; Wild Oryza species in serum content 1 ± 0.2% (v/v);
10-14 hour before initial cultivation, with half volume culture medium culturing;
Further, controlling dissolved oxygen is 40 ± 10% (v/v); Control ph is respectively 7.2 ± 0.2;
(3) acquisition mesenchymal stem cells MSCs is separated.
2. the method for claim 1, it is characterized in that, in step (1), the method for described low serum domestication is: joined successively by mesenchymal stem cells MSCs in the substratum that serum content successively decreases successively, make mesenchymal stem cells MSCs adapt to low serum environment.
3. method as claimed in claim 2, it is characterized in that, described low serum acclimation method is:
(a) by cell with 1 × 10
5cell/ml density is seeded in the substratum of 10% (v/v) serum, cultivates 48h, gathers in the crops the cell converged close to 90%;
B () cell is again with 2 × 10
5cell/ml density is seeded in the substratum of 7% (v/v) serum, when Growth of Cells is to when converging close to 90%, and trysinization harvested cell, and be seeded to identical density in the substratum of 7% serum and cultivate;
C the cell of () etc. (b) regrows to close to when converging, the flow process of the cell of results repetition (b) be seeded to successively in the substratum of 5% (v/v), 3% (v/v), 1% (v/v) serum and cultivate.
4. the method for claim 1, is characterized in that, changes substratum 50% every 0.5-2 days.
5. the method for claim 1, is characterized in that, in step (2), within first 12 hours, carries out intermittent stirring, 3min/h; Within 13-48 hour, cultivate with 35rpm rotating speed; Within 49-96 hour, cultivate with 45rpm rotating speed; 97 hours and cultivate with 50rpm rotating speed afterwards.
6. the method for claim 1, is characterized in that, described substratum is DMEM/F12 substratum.
7. the method for claim 1, it is characterized in that, in step (3), being separated the method obtaining mesenchymal stem cells MSCs is: from substratum, be separated the microcarrier cytodex3 having attached cell, washing, with tryptic digestion, interval concussion, to make cell come off from microcarrier cytodex3, is separated and removes microcarrier cytodex3.
8. be prepared into a chondrocyte's method, it is characterized in that, described method comprises:
I () obtains mesenchymal stem cells MSCs according to the arbitrary described method of claim 1-7;
(ii) mesenchymal stem cells MSCs of (i) is carried out chondroblast differentiation-inducing, thus obtain chondroblast.
9. prepare an osteoblastic method, it is characterized in that, described method comprises:
I () obtains mesenchymal stem cells MSCs according to the arbitrary described method of claim 1-7;
(ii) mesenchymal stem cells MSCs of (i) is carried out scleroblast differentiation-inducing, thus obtain scleroblast.
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| CN106011066A (en) * | 2016-07-07 | 2016-10-12 | 温州生物材料与工程研究所 | Method for large-scale preparation of human PC-3 cells |
| CN107674859B (en) * | 2017-08-28 | 2021-01-05 | 浙江大学 | Method for inducing mouse fibroblast to form cartilage by using small molecule composition |
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