CN1994478A - Method for amplifying mensenchymal stem cell under three-dimensional dynamic condition - Google Patents
Method for amplifying mensenchymal stem cell under three-dimensional dynamic condition Download PDFInfo
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Abstract
The invention relates to a method for expanding the stem cell between marrows in three-dimension dynamic condition, wherein it is characterize in that: hollow fiber film uses I-type collagen gel to embed the marrow stem cell; then cultivates said cell in cyclone biological reactor to obtain the marrow stem cell. The invention has the advantages that: I-type collagen gel can provide better three-dimension growth condition for stem cell, to cultivate cell multilayer; the hollow fiber film can protect cell from flow shearing force and impact; the cyclone reactor can reduce the transmission resistance of cultivate system, to provide enough nourishment to cell.
Description
Technical field
The invention belongs to biotechnology and field of tissue engineering technology, particularly a kind of method of amplifying mensenchymal stem cell under three-dimensional dynamic condition.
Background technology
Seed cell is the most basic raw material of organizational project, and a spot of cell is formed the tissue with certain 26S Proteasome Structure and Function through cultivating, increase and planting on extracellular matrix, is most important link in the Tissue Engineering Study.But most of histiocytic sources are limited, and separation difficulty has limited the progress of Tissue Engineering Study.
Mesenchymal stem cells MSCs is external draws materials easily, can height self duplication and renewal.It not only has potential (the Pittenger MF et al. to a plurality of direction differentiation such as osteoblast, chondrocyte, myocardial cell, adipose cell, fibroblast, skein cell, Science, 1999,284 (1)), and when body is subjected to wound, aging, disease equivalent damage, these cells will proliferation and differentiation, produces new tissue and substitutes them, to keep the homeostasis of body.Mesenchymal stem cells MSCs also has critical function (the Ueda Tet al. of hematopoiesis support, J Clin Invest, 2000,105:1013-1021), the recovery and reconstruction (the Almeida-Porada G et al. that can quicken patient's hemopoietic function with the hematopoietic stem cell co-transplantation, Blood 2000,95:3620-3627; Noort WA et al., Exp Hematol 2002,30:870-878; Angelopoulou M, et al., ExpHematol 2003,31:413-420), a difficult problem of capturing the hematopoietic stem cell transplantation treatment clinically are significant.In addition, mesenchymal stem cells MSCs is implanted a little less than the reaction, be easy to by exogenous gene transfection and stably express, be considered to ideal target cell (Suzanne Kadereit in organizational project, cell and the gene therapy, Adult Stem Cells (PhD), International Society for Stem Cell Research).Particularly some growth course has the disease of genetic defect, it is very big often to treat difficulty, as some muscular dystrophy, osteodysplasty etc., from normal donor, separate mesenchymal stem cells MSCs or the external evoked differentiation of process, implant ill receptor then, then be expected to improve the function of receptor linked groups.Therefore, mesenchymal stem cells MSCs is subjected to paying close attention to widely and paying attention to just as a kind of novel seed cell.
Cultivation and amplification about mesenchymal stem cells MSCs at present mainly contains static and dynamic two kinds of methods.Static state mainly comprises monolayer plate culture (Friedenstein AJ et al., Cell Tissue Kinet, 1987,20 (3)) and in culture plate, adopt type i collagen (Kiyoshi Yoneno et al., Journal of Biomedicalmaterials Research Part A, 2005,75A (3)), or type i collagen and agarose complex (AnnaBatorsky et al., Biotechnology and Bioengineering, 2005,92 (4)), fibrin complex (Shimony N et al., Kidney International, 2006,69 (3)) or alginate (Markusen JF, etal., Tissue Engineering, 2006,12 (4)) isogel is as the dimensional culture method of carrier; Dynamically cultivation mainly is to utilize microcarrier (Qin Q et al., Tissue Engineering, 1998,4 (1) in bioreactor; Glowacki J et al., Cell Transplant, 1998,7 (3)) carry out dimensional culture, or with mesenchymal stem cells MSCs and hematopoietic stem cell (Xi Chen et al. in rotating wall vessel bioreactor, Stem Cells, 2006, published online May25) carrying out three-dimensional cultivates altogether.
Static culture easy operating and control, but unit are inner cell amplification limited amount during monolayer culture, the requirement of the seed cell enormous amount that can't meet clinical needs; Though the cells expanded that dimensional culture obtains is than the increase to some extent of monolayer culture, especially type i collagen itself is exactly a kind of extracellular matrix, as the tissue support thing, closely related with reparation of growth, differentiation, hypertrophy and the tissue injury of cell etc., be very beneficial for the propagation of cell.But because nutrient substance is subjected to the influence of resistance to mass tranfer under static conditions, can not bring into play the function of cultivating system to greatest extent because of can not get enough nutrient apoptosis, cause the wasting of resources attached to the cell of carrier inside.
The dynamic 3 D cultivating system can be cell and provides and more be bordering on intravital three-dimensional environment, and no matter cell is can overcome contact inhibition on the microcarrier or in rotation wall type cultivating system, form the multilamellar growth, and cell amplification quantity obviously increases.But seed cell attached to the microcarrier surface, can sustain damage because of the mutual collision between shearing force and microcarrier, microcarrier is to the absorption of culture fluid composition and metabolite in addition, cause cell aggregation easily, cause cell differentiation (Yoo JU et al., J Bone Joint Surg Am, 1998,80 (12); Johnstone B et al., Clin Orthop, 1999, (367 Suppl): S156), reduced the quality of seed cell.Though rotation wall type cultivating system shearing force is lower, has reduced the damage that the shearing force pair cell causes, and has certain Concentraton gradient, the supply of nutrient is inhomogeneous.Therefore, need the suitable Three-Dimensional Dynamic cultivating system of development, improve culture environment to turn out all guaranteed mesenchymal stem cells MSCs of quality and quantity.
Summary of the invention
The purpose of this invention is to provide the method for cultivating under a kind of Three-Dimensional Dynamic condition with the amplification mesenchymal stem cells MSCs.
Technical scheme of the present invention may further comprise the steps:
1. mesenchymal stem cells MSCs is provided.Get the new zealand white rabbit about body weight 2.5kg in age all around, press 1mLkg with 3% pentobarbital sodium
-1After the intravenous injection anesthesia, be needled into the rabbit femoral medullary cavity with bone marrow aspiration under the aseptic condition, connect the 10mL syringe, include 3000UmL
-1Heparin 0.5mL, extract bone marrow 4-6mL out, after low sugar serum-free DMEM dilution, with proportion be 1.073gmL
-1The Percoll separating medium carry out gradient centrifugation by 1: 1 volume ratio, the centrifugal 25min of 3000rpm, interface milky cloud suspension cell is to another centrifuge tube, with low sugar serum-free DMEM washed twice, the centrifugal 5min of 1200rpm in the middle of slowly drawing.Remove supernatant, add the DMEM culture medium that contains 20% hyclone, adjust cell density, with 5 * 10
5CellsmL
-1Density be inoculated in the culture bottle.Remove suspension cell after three days, per five and half amounts are changed liquid.When former generation the rabbit bone marrow mescenchymal stem cell reaching 80% at the bottom of the culture bottle when merging, adding and containing 0.02%EDTA concentration is 0.25% trypsinization, in the cultivation of going down to posterity of 1: 3 ratio.
2. preparation hollow-fibre membrane.It is after hydrophilicity kynoar (PVDF) hollow-fibre membrane of 0.1 μ m cleans repeatedly that internal diameter and external diameter are respectively 0.8mm and 1.4mm, wall aperture, and 121 ℃ of sterilization 30min are stand-by.
3.I Collagen Type VI gel embedding mesenchymal stem cells MSCs.To contain 3ngmL
-1BFGF concentration is 3mgmL
-1Type i collagen solution add in the rabbit bone marrow mesenchyma stem cell suspension of certain cell quantity, adjusting cell density is 5 * 10
5CellsmL
-1, be inoculated in the Kynoar hollow-fibre membrane, put 37 ℃, 5%CO
2, saturated humidity CO
2In film, form the gel that contains cell in the incubator after 2 hours.
4. preparation airlift loop bioreactor.Before the experiment, (air-lift loop bioreactor, behind each parts ALB), ultra-pure water washes three times to clean airlift loop bioreactor with abluent and soft brush.Assemble the main part of ALB, main part and the pipeline that connects are wrapped up, 121 ℃ of sterilization 30min, dry for standby.
5. in airlift loop bioreactor, Three-Dimensional Dynamic is cultivated mesenchymal stem cells MSCs.Assembling ALB cell culture system under the aseptic condition in super-clean bench is seeded to the type i collagen gel film assembly of embedding mesenchymal stem cells MSCs in the reactor, adds and contains 20% hyclone, 3ngmL
-1The DMEM culture medium of bFGF is put the ALB cell culture system 37 ℃, 5%CO then
2, saturated humidity CO
2In the incubator, open air pump 2 switches, the adjustments of gas flow makes culture fluid fully circulation in circulation bioreactor.
6. the enzymolysis gel is gathered in the crops mesenchymal stem cells MSCs.After cultivating end, take out the type i collagen gel film assembly of embedding mesenchymal stem cells MSCs, hollow-fibre membrane is inserted in the centrifuge tube, and adding concentration is 2.5% type i collagen enzyme, 37 ℃ of following enzymolysis 20min, the centrifugal 5min of 1200rpm, abandon supernatant, add the 1mL culture fluid, piping and druming, the meter cell number is seeded in the culture bottle and cultivates.
Effect of the present invention and advantage are:
(1) mesenchymal stem cells MSCs is dynamically cultivated under three-dimensional condition, effectively be reduced the resistance to mass tranfer of cultivating system, make cell have competent nutrient supply, cell amplification quantity obviously increases.
(2) timbering material type i collagen gel more is bordering on intravital three-dimensional environment for cell provides, and has promoted the contact between the cell, makes cell form the multilamellar growth in gel.
(3) grow in the gel of cell in hollow-fibre membrane, avoided the effect of film outer fluid shearing force fully, and avoided cell collision each other, thus the damage of having avoided common Airlift circulating reactor pair cell to be brought, this cultivates particularly important for stem cell.
(4) utilize the method for a kind of amplifying mensenchymal stem cell under three-dimensional dynamic condition of the present invention, the rabbit bone marrow mescenchymal stem cell of type i collagen gel embedding in the hollow-fibre membrane has been carried out the Three-Dimensional Dynamic cultivation.The result shows that cell is well-grown in this cultivating system, mononuclearcell, CD44 after seven days
+CD45
-The amplification times of cell can reach 20 times and 31 times respectively.
Description of drawings
Fig. 1 is the method operational flowchart of a kind of amplifying mensenchymal stem cell under three-dimensional dynamic condition of the present invention.
Fig. 2 is the hollow fiber film structure sem photograph of a kind of amplifying mensenchymal stem cell under three-dimensional dynamic condition of the present invention.
Fig. 3 is the process chart of Airlift circulating cultivating system of the method for a kind of amplifying mensenchymal stem cell under three-dimensional dynamic condition of the present invention.
Fig. 4 is the method for a kind of amplifying mensenchymal stem cell under three-dimensional dynamic condition of the present invention, the microphotograph of type i collagen gel embedding rabbit bone marrow mescenchymal stem cell.
Fig. 5 is the method for a kind of amplifying mensenchymal stem cell under three-dimensional dynamic condition of the present invention, the microphotograph (A:Day1 that the rabbit bone marrow mescenchymal stem cell is cultivated in the type i collagen gel under the static conditions; B:Day7).
Among the figure: 1.CO
2Incubator; 2. air pump; 3. spinner flowmeter; 4. gas filtration film; 5. hollow fiber film assembly; 6. froth breaking filler; 7. support.
The specific embodiment
Be described in detail specific embodiments of the invention below in conjunction with technical scheme and accompanying drawing.
Embodiment 1:
Present embodiment be under the static conditions rabbit bone marrow mesenchyme in the cultivation of cell in the type i collagen gel.
For verify cell can be in the type i collagen gel well growth, adopt type i collagen gel embedding rabbit bone marrow mescenchymal stem cell, carry out In vitro culture and amplification.Particularly, be 10 * 10 with cell number
5The rabbit bone marrow mescenchymal stem cell of cells with contain 3ngmL
-1BFGF concentration is 3mgmL
-1Type i collagen solution 1mL mix homogeneously, be seeded in 96 well culture plates every hole 50 μ L.Put 37 ℃, 5%CO
2, saturated humidity CO
2Formed the gel that contains cell in 2 hours in the incubator, add and contain 20% hyclone, 3ngmL
-1The DMEM culture fluid 100 μ L of bFGF, the density of cell this moment in every hole is 5 * 10
5CellsmL
-1, put 37 ℃, 5%CO
2, saturated humidity CO
2Cultivate in the incubator.The mesenchymal stem cells MSCs suspension direct inoculation that other gets a same cell density to the culture bottle in contrast group carry out static culture.
Cultivate after 7 days, with the type i collagen gel of embedding mesenchymal stem cells MSCs with 37 ℃ of following enzymolysis 20min of type i collagen enzyme of 2.5%, the centrifugal 5min of 1200rpm, abandon supernatant, add the 1mL culture fluid, piping and druming, counting is induced to osteoblast, chondrocyte and adipose cell direction respectively; Flow cytometer detects CD44
+CD45
-Cell number.Gather in the crops the mesenchymal stem cells MSCs of matched group simultaneously, digestion, piping and druming, counting is induced to osteoblast, chondrocyte and adipose cell respectively; Flow cytometer detects CD44
+CD45
-Cell number.
More than test equal triplicate.
Experimental result: cultivate after 7 days, cellular control unit density reaches 15 * 10
5CellsmL
-1, amplification times is 3 times; Type i collagen gel embedding group cell density reaches 35 * 10
5CellsmL
-1, amplification times is 7 times.Induce differentiated result to show, the resulting cell of experimental group and matched group can both be to osteoblast, chondrocyte and adipose cell differentiation; The flow cytometer testing result is found experimental group and matched group CD44
+CD45
-The cell percentage composition is mesenchymal stem cells MSCs all above 70%.Microphotograph (the A:Day1 that Fig. 4 cultivates in the type i collagen gel for rabbit bone marrow mescenchymal stem cell under the static conditions; B:Day7).
This embodiment explanation: mesenchymal stem cells MSCs can well be grown in the type i collagen gel; Under the static culture conditions, in the type i collagen gel cultured cells amplification times obviously more than common cultured cells, simultaneous verification the mass-transfer performance of type i collagen gel good; Especially stem cell is very responsive to shearing force owing to zooblast, and prompting adopts the dynamic cultured cells of type i collagen gel may breed more under the situation of avoiding the shearing force damage.
Embodiment 2:
The yield rate of cell when present embodiment is type i collagen gel embedding rabbit bone marrow mescenchymal stem cell enzymolysis collagen.
Adopt 3mgmL
-1Type i collagen gel embedding rabbit bone marrow mescenchymal stem cell, determine the yield rate of cell by type i collagen enzyme enzymolysis type i collagen.Particularly, be 25 * 10 with cell number
5Cells rabbit bone marrow mescenchymal stem cell and 0.5mL concentration are 3mgmL
-1Type i collagen solution mix homogeneously, be seeded in 96 orifice plates, every hole 50 μ L put 37 ℃, 5%CO
2, saturated humidity CO
2Formation in 2 hours contains the gel of cell in the incubator.37 ℃ of following enzymolysis 20min of type i collagen enzyme of adding 2.5%, the centrifugal 5min of 1200rpm abandons supernatant, adds the 1mL culture fluid, piping and druming, counting calculates the cell harvesting rate.
More than test triplicate.
Experimental result: three times experiment gained cell harvesting rate is respectively 88.7%, 94.2%, 90.4%, and average yield rate reaches 91.1%.This embodiment explanation: it is feasible adopting the method for type i collagen enzyme enzymolysis type i collagen harvesting; Behind cell culture, can gather in the crops most of expanded cells by this method.Fig. 5 is 3mgmL
-1The microphotograph of type i collagen gel embedding rabbit bone marrow mescenchymal stem cell.
Embodiment 3:
Present embodiment is the cultivation of type i collagen gel embedding rabbit bone marrow mescenchymal stem cell in airlift loop bioreactor in the hollow-fibre membrane.
In order to verify successfully culturing stem cells of ALB cell culture system, present embodiment is cultivated the rabbit bone marrow mescenchymal stem cell of type i collagen gel embedding in the hollow-fibre membrane in the ALB culture systems.
The separation of rabbit bone marrow mescenchymal stem cell: get the new zealand white rabbit about body weight 2.5kg in age all around, press 1mLkg with 3% pentobarbital sodium
-1After the intravenous injection anesthesia, under the sterile working, be needled into the rabbit femoral medullary cavity, connect the 10mL syringe, include 3000UmL with bone marrow aspiration
-1Heparin 0.5mL, extract bone marrow 4-6mL out, after low sugar serum-free DMEM dilution, with proportion be 1.073gmL
-1The Percoll separating medium carry out gradient centrifugation by 1: 1 volume ratio, the centrifugal 25min of 3000rpm, interface milky cloud suspension cell is to another centrifuge tube, with low sugar serum-free DMEM washed twice, the centrifugal 5min of 1200rpm in the middle of slowly drawing.Remove supernatant, add the DMEM culture medium that contains 20% hyclone, adjust cell density, with 5 * 10
5CellsmL
-1Density be inoculated in the culture bottle.Remove suspension cell after three days, per five and half amounts are changed liquid.When former generation the rabbit bone marrow mescenchymal stem cell reaching 80% at the bottom of the culture bottle when merging, adding and containing 0.02%EDTA concentration is 0.25% trypsinization, in the cultivation of going down to posterity of 1: 3 ratio.
The preparation of hollow-fibre membrane before the experiment: after the hydrophilicity kynoar hollow-fibre membrane that internal diameter and external diameter are respectively 0.8mm and 1.4mm cleans repeatedly, 121 ℃ of 30min that sterilize, stand-by.
Type i collagen gel embedding rabbit bone marrow mescenchymal stem cell: will contain 3ngmL
-1BFGF concentration is 3mgmL
-1Type i collagen solution add in the rabbit bone marrow mesenchyma stem cell suspension of certain cell quantity, adjusting cell density is 5 * 10
5CellsmL
-1, be inoculated in the Kynoar hollow-fibre membrane, put 37 ℃, 5%CO
2, saturated humidity CO
2In film, form the gel that contains cell in the incubator after 2 hours.
The preparation of airlift loop bioreactor before the experiment: before the experiment, behind each parts with abluent and soft brush cleaning ALB, ultra-pure water flushing three times.Assemble the main part of ALB, main part and the pipeline that connects are wrapped up, 121 ℃ of sterilization 30min, dry for standby.
The cultivation of mesenchymal stem cells MSCs in airlift loop bioreactor: assembling ALB cell culture system under the aseptic condition in super-clean bench, the type i collagen gel film assembly of embedding mesenchymal stem cells MSCs is seeded in the reactor, adds and contain 20% hyclone, 3ngmL
-1The DMEM culture medium of bFGF is put the ALB cell culture system 37 ℃, 5%CO then
2, saturated humidity CO
2In the incubator, open air pump switch, the adjustments of gas flow makes culture fluid fully circulation in circulation bioreactor.
Cultivate after 7 days, take out the type i collagen gel film assembly of embedding mesenchymal stem cells MSCs, hollow-fibre membrane is inserted in the centrifuge tube, and adding concentration is 2.5% type i collagen enzyme, 37 ℃ of following enzymolysis 20min, the centrifugal 5min of 1200rpm, abandon supernatant, add the 1mL culture fluid, piping and druming, counting is induced to osteoblast, chondrocyte and adipose cell respectively; Flow cytometer detects CD44
+CD45
-Cell number.
Cultivation results: after the rabbit bone marrow mescenchymal stem cell of type i collagen gel embedding was cultivated 7 days in the hollow-fibre membrane in airlift loop bioreactor, mononuclearcell had increased 20 times, CD44
+CD45
-Cell amplification more than 30 times, and can be induced to differentiate into osteoblast, chondrocyte and adipose cell.
This embodiment explanation: airlift loop bioreactor can effectively reduce the resistance to mass tranfer of cultivating system, makes cell have competent nutrient supply; Type i collagen for mesenchymal stem cells MSCs provides good 3 D stereo growing environment, has promoted the contact between the cell as extracellular matrix, makes cell form the multilamellar growth, and cell amplification quantity obviously increases; Grow in the gel of cell in hollow-fibre membrane, avoided the effect of film outer fluid shearing force fully, and avoided cell collision each other, thereby the damage of having avoided common Airlift circulating reactor pair cell to be brought has improved the culture effect of cell.It is feasible using this reactor In vitro culture and amplification purpose cell.
Although the present invention is that example is described with type i collagen gel embedding rabbit bone marrow mescenchymal stem cell in the hollow-fibre membrane, this description and not meaning that is construed as limiting the present invention.With reference to description of the invention, other distortion of the cell of other kind, gel, hollow-fibre membrane and embodiment all can be expected for those skilled in the art.Therefore, such distortion can not break away from affiliated claim restricted portion and spirit.
Claims (1)
1. the method for an amplifying mensenchymal stem cell under three-dimensional dynamic condition is characterized in that following steps:
(1) provide mesenchymal stem cells MSCs: the mesenchymal stem cells MSCs in rabbit femoral source is with 5 * 10
5CellsmL
-1Density be inoculated in the DMEM culture medium that contains 20% hyclone and cultivate, when cell is reaching 80% when merging at the bottom of the culture bottle, adding and containing 0.02%EDTA concentration is 0.25% trypsinization, carries out the routine cultivation of going down to posterity in 1: 3 ratio;
(2) prepare hollow-fibre membrane: after the hydrophilicity kynoar hollow-fibre membrane that internal diameter and external diameter are respectively 0.8mm and 1.4mm cleans repeatedly, 121 ℃ of 30min that sterilize;
(3) type i collagen gel embedding mesenchymal stem cells MSCs: will contain 3ngmL
-1BFGF concentration is 3mgmL
-1Type i collagen solution add in the rabbit bone marrow mesenchyma stem cell suspension of certain cell quantity, adjusting cell density is 5 * 10
5CellsmL
-1, be inoculated in the Kynoar hollow-fibre membrane, put 37 ℃, 5%CO
2, saturated humidity CO
2Interior 2 hours of incubator forms the gel that contains cell in film;
(4) prepare airlift loop bioreactor: with each parts of abluent and soft brush cleaning airlift loop bioreactor, ultra-pure water flushing three times; Assemble the main part of reactor, main part and the pipeline that connects are wrapped up, 121 ℃ of sterilization 30min, oven dry;
(5) in airlift loop bioreactor, Three-Dimensional Dynamic is cultivated mesenchymal stem cells MSCs: the type i collagen gel film assembly of embedding mesenchymal stem cells MSCs is seeded in the airlift loop bioreactor, adds and contain 20% hyclone, 3ngmL
-1The DMEM culture medium of bFGF is put 37 ℃, 5%CO
2, saturated humidity CO
2In the incubator, open air pump switch, the adjustments of gas flow makes culture fluid fully circulation in circulation bioreactor;
(6) enzymolysis gel, the results mesenchymal stem cells MSCs: employing concentration is 37 ℃ of following enzymolysis type i collagen gel 20min of type i collagen enzyme of 2.5%, the centrifugal 5min of 1200rpm, results mesenchymal stem cells MSCs.
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