CN101698830B - Method for in-vitro culture and virus transfection of bone marrow mesenchymal stem cells of adult crab eating monkeys - Google Patents
Method for in-vitro culture and virus transfection of bone marrow mesenchymal stem cells of adult crab eating monkeys Download PDFInfo
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Abstract
The invention relates to a method for in vitro culture and virus transfection of bone marrow mesenchymal stem cells for adult crab eating monkeys. In the method disclosed by the invention, an antiviral medicament is added in an in-vitro culture medium for the bone marrow mesenchymal stem cells of the adult crab eating monkeys so as to increase the number of in-vitro passages for the bone marrow mesenchymal stem cells of the adult crab eating monkeys and to increase the cell amplification number. In the invention, the cell amplification number is increased in a large scale by further improving the culture condition. The virus transfection efficiency for the bone marrow mesenchymal stem cells of the adult crab eating monkeys of in vitro culture is enhanced by improving the method for performing virus transfection and in vitro culture on the bone marrow mesenchymal stem cells of the adult crab eating monkeys.
Description
Technical field
The present invention relates to the vitro culture of mesenchymal stem cells MSCs and to the method for its virus transfection, relating more specifically to infect has the bone marrow mesenchymal stem cells of adult crab eating monkeys of MK virus vitro culture and to the method for its virus transfection.
Background technology
Mescenchymal stem cell (mesenchymal stem cell; MSC) be the stem cell with multidirectional differentiation capability in mesoderm source; Mainly be present between whole body reticular tissue and organ in the matter; The abundantest with content in the myeloid tissue, be adult stem cell with self-replacation, multidirectional differentiation potential.Under certain inductive condition, has ability to the differentiation of mesoblastemas such as scleroblast, chondroblast, sarcoplast, Tenocyte cell, adipocyte and stroma cell; Simultaneously, MSC can also be to ectodermic neuronal cell and the differentiation of endoblastic liver ovum circle sexual cell.This has broken through the notion that MSC is merely mesodermal stem cell, has disclosed MSC and has even more important theoretical significance and more wide application prospect.Marrow MSC in-vitro separation is cultivated and is obtained easily, and cell immunogenicity is low, is easy to stablized by exogenous virus transfection and expression, has become ideal seed cell in organizational project, gene therapy and the regenerative medicine research.The application of mescenchymal stem cell is to carry out through the direct autotransplantation of cell or cell expansion ex vivo and autoplastic mode mostly.
No matter be to utilize marrow MSC as transplanted cells or engineered carrier cell, the method for its treatment or use and effect and curative effect all need be passed through a large amount of experiments and verified.But carry out the restriction that scientific research receives many factors with the patient for direct research object, the restriction of especially ethics aspect.Therefore, need at first on the body of laboratory animal, to confirm the curative effect of various treat-ment.
In all laboratory animal, generally believe that primate mammal is and the immediate animal of the mankind.Cynomolgus monkey is as a kind of extensive in recent years monkey section primate mammal of raising, and in before various treat-ment and medicine clinical, testing, bringing into play more and more important effect.But in research to some treatment of diseases method; Again must be with the primate mammal of growing up; Especially adult crab eating monkeys is an object, like some diseases that in grownup and the elderly, just take place such as senile dementias, only with the primate mammal of growing up; Especially adult crab eating monkeys is the Transplanted cells after research object is carried out virus transfection, and the experimental data of acquisition is just more valuable.
In the application that with the cynomolgus monkey is being object research mesenchymal stem cells MSCs, we find that the marrow MSC of adult crab eating monkeys is very limited at subculture in vitro separately algebraically, and cell merges very soon, in the marrow MSC of external cynomolgus monkey be difficult to the to increase quantity of needs.Only large stretch of cytogamy just appears to the marrow MSC vitro culture of adult crab eating monkeys in 3 generations.Can't carry out the amplification of cell quantity and external genetic engineering modified experiment.
Summary of the invention
One of the object of the invention is through to growing up primate mammal; Especially the improvement of the condition of in vitro culture of bone marrow mesenchymal stem cells of adult crab eating monkeys; Improve the primate mammal of growing up significantly; Especially bone marrow mesenchymal stem cells of adult crab eating monkeys is in the amplification quantity of vitro culture, so that cell quantity more is prone to reach the amount that needs.
Another object of the present invention is through the improvement to transfection conditions, thereby the raising slow virus is to the transfection efficiency of grow up primate mammal, especially bone marrow mesenchymal stem cells of adult crab eating monkeys.
The present invention provides a kind of vitro culture primate mammal of growing up; Especially the method for bone marrow mesenchymal stem cells of adult crab eating monkeys; Comprise: I) to the mesenchymal stem cells MSCs from adult primate mammal, especially adult crab eating monkeys carry out former be commissioned to train foster; II) when cell grows into 70%～80% degree of converging, digest, go down to posterity and cultivate; III) when the said cultured cells that goes down to posterity grows into 70%～80% degree of converging, digest once more, go down to posterity and cultivate; What the said cultivation of going down to posterity was used is basic medium, in said basic medium, is added with foetal calf serum, penicillium mould and Streptomycin sulphate; It is characterized in that: also be added with antiviral in the said basic medium of cultivating that goes down to posterity.
A kind of preferred embodiment in, said antiviral is antiretroviral drugs preferably.
A kind of preferred embodiment in, said antiretroviral drugs is tynofovir preferably.
A kind of preferred embodiment in, the preferred mixture that adds FGF-b or FGF-b and EGF in said basic medium.
A kind of preferred embodiment in, said FGF-b or said EGF be preferably recombinant human FGF-b or recombinant human EGF respectively.
A kind of preferred embodiment in, also add the GlutaMAX-I dipeptides in the preferred said basic medium, said basic medium is α lpha modified version MEM substratum preferably.
A kind of preferred embodiment in, said foetal calf serum is the special-purpose foetal calf serum of mesenchymal stem cells MSCs preferably.
A kind of vitro culture primate mammal of growing up; Especially the method for bone marrow mesenchymal stem cells of adult crab eating monkeys and external virus transfection; Comprise: I) to the mesenchymal stem cells MSCs from adult primate mammal, especially adult crab eating monkeys carry out former be commissioned to train foster; II) when cell grows into 70%～80% degree of converging, digest, go down to posterity and cultivate; III) when the said cultured cells that goes down to posterity grows into 70%～80% degree of converging, digest once more, go down to posterity and cultivate; IV) when cell grow into 3-5 for the time, said adult primate mammal, especially bone marrow mesenchymal stem cells of adult crab eating monkeys are carried out virus transfection; What the said cultivation of going down to posterity was used is basic medium, in said basic medium, is added with foetal calf serum, penicillium mould and Streptomycin sulphate and antiviral; It is characterized in that: carrying out step IV) virus transfection before the said basic medium that will be added with foetal calf serum, penicillium mould and Streptomycin sulphate and antiviral in 2-6 days be replaced by the basic medium that is added with foetal calf serum, penicillium mould and Streptomycin sulphate that does not contain antiviral.
A kind of preferred embodiment in, said antiviral is antiretroviral drugs preferably.
A kind of preferred embodiment in, said antiretroviral drugs is tynofovir preferably.
A kind of preferred embodiment in, said virus is slow virus preferably.
Use method of the present invention, said adult primate mammal, especially bone marrow mesenchymal stem cells of adult crab eating monkeys make the algebraically that cell can go down to posterity increase after cultivating, and the feasible amplification cell quantity showed increased that can access.
Use the adult primate mammal of vitro culture provided by the invention; Especially bone marrow mesenchymal stem cells of adult crab eating monkeys and to the method for its external virus transfection; Can obviously improve the transfection efficiency of virus to grow up primate mammal, especially bone marrow mesenchymal stem cells of adult crab eating monkeys.
Description of drawings
Figure 1A and Figure 1B are respectively embodiments of the invention 1 photo when growing into the s-generation with cell in the Comparative Examples 1, and microscopical magnification is 100 times among Figure 1A, is 200 times among Figure 1B.
Fig. 2 is the broken line graph that the cell quantity of cell after external amplification the in embodiments of the invention 1 and the Comparative Examples 1 increases.
Fig. 3 is the column map that the cell quantity of cell after external amplification the in embodiments of the invention 2,3 and the Comparative Examples 2 increases.
Fig. 4 is the column map that the cell quantity of cell after external amplification the in embodiments of the invention 4 and the Comparative Examples 3,4,5 increases.
Fig. 5 is the column map that the cell quantity of cell after external amplification the in embodiments of the invention 5 and the Comparative Examples 6 and 7 increases.
Fig. 6 broken line graph that to be embodiments of the invention 6 increase with the cell quantity of cell after external amplification the in the Comparative Examples 8, ◆ represent cell among the embodiment 6 in the quantity of amplification in vitro, ▲ represent cell in the Comparative Examples 8 in the quantity of amplification in vitro.
The cell that Fig. 7 shows in embodiments of the invention 7 and the Comparative Examples 9 is being after carrier carries out transfection with the slow virus, expresses the proteic positive cell percentage of GFP.
The cell that Fig. 8 shows in embodiments of the invention 7 and the Comparative Examples 9 is being after carrier carries out transfection with the slow virus; Viewed cell under fluorescent microscope; Microscopical magnification is 200 times in Fig. 8 B and 8G, and magnification is 100 times in Fig. 8 D and 8I.
Fig. 9 shows the gene constructed figure (9A) of the packaging plasmid CMV Δ 8.91 that uses among the present invention, the gene constructed figure (9B) of packaging plasmid PMD.G, the gene constructed figure (9C) of vector plasmid Duet101.
Embodiment
In experiment of the present invention, used the adult crab eating monkeys in 6 8-10 years, the infection rate of its MK virus is 100%.These adult crab eating monkeys are all from Guangxi Nanning Lingkang Senoke Biology Science and Technology Co., Ltd; The animal facility of the said firm has obtained International Laboratory Animal management assessment and IPCA (Association for Assessment and Accreditationof Laboratory Animal Care, qualification certification AAALAC).In the present invention, the mesenchymal stem cells MSCs of growing up is from adult crab eating monkeys, and these adult mesenchymal stem cells MSCs can be that following steps are said and take from adult crab eating monkeys alive, can certainly be to take from the animal body of just having put to death.The process of getting bone marrow mesenchymal stem cells of adult crab eating monkeys is following:
1) with bone puncture needle, gauze, operation drape sterilization;
2) ketamine (15mg/kg) intramuscular injection, Animal Anesthesia;
3) coromegine (0.04mg/kg) intramuscular injection reduces secretory product;
4) preserved skin, sterilization, shop operation towel, confirm posterior superior iliac spine, with lignocaine (2-3ml) toponarcosis;
5) regulate bone puncture needle, bone puncture needle is vertical with surface of bone to puncture, and fixedly puncture needle is in ilium;
6) extract nook closing member, extract 5-10ml marrow, mixing with the syringe that contains heparinized saline;
7) extract puncture needle, after 3-5min is pushed in the sterile gauze part, add press fit with gauze.Postoperative uses Kefzol (25mg/kg) intravenous drip, preventing infection;
8), use the DPBS dilution of volume, mixing as marrow volume twice according to the amount of taking out marrow;
9) in the 15ml centrifuge tube, adding 3ml density is 1.073 Ficoll-PaqueTMPlus parting liquid (Stem Cell company);
10) slowly add the marrow after 6ml dilutes with the pasteur pipe, on the Ficoll-PaqueTMPlus parting liquid that is added to gently;
11) 2500rpm is centrifugal 30 minutes, is divided into 4 layers in the centrifuge tube, is respectively serum, monocyte, Ficoll-PaqueTMPlus parting liquid and red corpuscle from top to bottom;
12) draw centrifuge tube intermediary mononuclear cell layer with the pasteur pipe, be transferred to new 15ml centrifuge tube;
13) according to the monocyte suspension volume of drawing, add DPBS according to 1: 3～1: 5 volume ratio, mixing, the centrifugal 10min of 1500rpm discards the liquid on upper strata, so washes cell twice;
14) with the monocyte suspension inoculation that obtains, containing 5%CO
2, cultivate after 3 days in 37 ℃ of incubators and change substratum;
15) cultivate 7-10 days, when cell length to 70%～80%, digest the cultivation of going down to posterity with 0.25%Trypsin (pancreatin)-EDTA.
The inventor finds that the mesenchymal stem cells MSCs of adult crab eating monkeys is in the process that subculture in vitro separately is cultivated; Be no more than the phenomenon that large stretch of fused cell will appear in 3 generations; This be because the mesenchymal stem cells MSCs of adult crab eating monkeys infected mostly MK virus (SimianFoamy Virus, SFV) (usually childhood cynomolgus monkey SFV infection rate be starkly lower than adult crab eating monkeys).Therefore; In the substratum that goes down to posterity of the mesenchymal stem cells MSCs of adult crab eating monkeys of the present invention, add antiviral, the phenomenon that can suppress cytogamy takes place, because MK virus is a kind of retrovirus; Therefore, the effect of interpolation antiretroviral drugs is better.In an embodiment of the present invention, through in the subculture in vitro separately substratum of bone marrow mesenchymal stem cells of adult crab eating monkeys, adding antiretroviral medicine,, above-mentioned phenomenon is disappeared like tynofovir.
The inventor makes cell increase in the quantity of amplification in vitro also through in the substratum that goes down to posterity, adding the mixture of FGF-b or FGF-b and EGF.Said FGF-b or said EGF can be respectively recombinant human FGF-b or recombinant human EGF.
The inventor is the basic medium through using α lpha modified version MEM substratum (α MEM) to go down to posterity and cultivate as the mesenchymal stem cells MSCs of adult crab eating monkeys also; And in α lpha modified version MEM substratum (α MEM), adding the GlutaMAX-I dipeptides, the result makes cell further increase in the quantity of amplification in vitro.
The inventor also through selecting for use the special-purpose foetal calf serum of mesenchymal stem cells MSCs to replace common foetal calf serum to add in the substratum that goes down to posterity of mesenchymal stem cells MSCs of adult crab eating monkeys, makes cell further increase in the quantity of amplification in vitro.
The cultivation of will going down to posterity from the mesenchymal stem cells MSCs of the cynomolgus monkey that grows up; In embodiment 1, in the substratum that goes down to posterity that the following basic medium that comprises other composition forms, add tynofovir (LGM pharmaceuticals), add 10 μ M in the substratum that goes down to posterity of 100ml; In comparative example 1, then do not add tynofovir.
Each composition that comprises in the substratum that goes down to posterity of 100ml: the DMEM of (1) 88% (volume percent) (invitrogen), this is a basic medium; (2) other composition: 10% excellent foetal calf serum (GIBCO), 1% L-glutaminate liquid (invitrogen), penicillium mould-Streptomycin sulphate liquid (invitrogen) of 1%.
The inoculation quantity that cell goes down to posterity for the first time is 1 * 10
5, the density of cell inoculation is 1000/cm
2
Cell among result: the embodiment 1 does not have very fast large stretch of phenomenon that merges that occurs, and can pass more than 10 generations.And large stretch of fusion phenomenon of cell has just appearred in the cell in Comparative Examples 1 in the 2nd generation, can not continue to go down to posterity.Can be referring to the cell photo among Fig. 1, and the broken line graph among Fig. 2.
In above experiment, can also use antiviral emtricitabine and Stocrin, other conditions are identical with embodiment 1, can reach the effect among the embodiment 1 equally.
Cultivations of will going down to posterity from the mesenchymal stem cells MSCs of the cynomolgus monkey that grows up, embodiment 2 with 3 and Comparative Examples 2 in the different growth factor of interpolation in the substratum that goes down to posterity respectively, in embodiment 2, add FGF-b (Invitrogen), concentration is 20ng/ml; In embodiment 3, add the mixture of FGF-b (Invitrogen) and EGF (Invitrogen), concentration respectively is 10ng/ml, and the total concn of two kinds of growth factors is 20ng/ml; In Comparative Examples 2, add EGF (Invitrogen), concentration is 20ng/ml.
Each composition of the substratum that goes down to posterity of 100ml is: the α MEM (Invitrogen) of (1) 88% (volume percent), and this is a basic medium; (2) the special-purpose foetal calf serum of other composition: 1%GlutaMAX-I (Invitrogen), 1% penicillium mould-Streptomycin sulphate liquid (Invitrogen), 10 μ M tynofovirs (LGM pharmaceuticals), 10% mesenchymal stem cells MSCs (GIBCO, AUS).
The inoculation quantity that cell goes down to posterity for the first time is 1 * 10
5, the density of cell inoculation is 1000/cm
2GlutaMAX-I is a kind of dipeptidase derivant of L-glutaminate, and with the protection of L-L-Ala, this dipeptidase derivant is highly stable with its unsettled alpha-amino group, and cracking at high temperature is less, and peptase can be with its cracking.
Cell amplification quantity among result: the embodiment 2 and 3 and speed are obviously greater than cell amplification quantity and speed in the Comparative Examples 2.Specifically referring to the histogram among Fig. 3.
Basic medium α MEM (Invitrogen) (volume percent is 88%) and interpolation factor GlutaMAX-I (Invitrogen) (volume percent is 1%) are used in cultivations of will going down to posterity from the mesenchymal stem cells MSCs of the cynomolgus monkey that grows up in embodiment 4; In Comparative Examples 3, use basic medium α MEM (Invitrogen) (volume percent is 88%) and L-glutaminate (Invitrogen) (volume percent is 1%); In Comparative Examples 4, use basic medium DMEM (volume percent is 88%) and GlutaMAX-I (Invitrogen) (volume percent is 1%); In Comparative Examples 5, use basic medium DMEM (volume percent is 88%) and L-glutaminate (Invitrogen) (volume percent is 1%).
Other compositions of the substratum that goes down to posterity of 100ml be the special-purpose foetal calf serum of 1% penicillium mould-Streptomycin sulphate liquid (Invitrogen), 10 μ M tynofovirs (LGM pharmaceuticals), 10% mesenchymal stem cells MSCs (GIBCO, AUS), concentration is the FGF-b (Invitrogen) of 20ng/ml.
The inoculation quantity that cell goes down to posterity for the first time is 1 * 10
5, the density of cell inoculation is 1000/cm
2
Cell amplification quantity among result: the embodiment 4 and speed are obviously greater than cell amplification quantity and speed in the Comparative Examples 3,4,5.Specifically referring to the histogram among Fig. 4.
Cultivations of will going down to posterity from the mesenchymal stem cells MSCs of the cynomolgus monkey that grows up, in embodiment 5, use MSC special use foetal calf serum (GIBCO, AUS); The excellent foetal calf serum that use USA produces in Comparative Examples 6 (invitrogen, USA); The excellent foetal calf serum that use AUS produces in Comparative Examples 7 (invitrogen, AUS).
Each composition of the substratum that goes down to posterity of 100ml is: (1) basic medium: the α MEM (Invitrogen) of 88% (volume percent); (2) other compositions: 1%GlutaMAX-I (Invitrogen), 1% penicillium mould-Streptomycin sulphate liquid (Invitrogen), 10 μ M tynofovirs (LGM pharmaceuticals), concentration are the FGF-b (Invitrogen) of 20ng/ml.
The inoculation quantity that cell goes down to posterity for the first time is 1 * 10
5, the density of cell inoculation is 1000/cm
2
Cell amplification quantity among result: the embodiment 5 and speed are obviously greater than cell amplification quantity and speed in Comparative Examples 6 and 7.Specifically referring to the histogram among Fig. 5.
The cultivation of will going down to posterity from the mesenchymal stem cells MSCs of the cynomolgus monkey that grows up; The medium component that in embodiment 6, uses is: and the α MEM (Invitrogen) of 88% (volume percent) (α MEM basic medium), the special-purpose foetal calf serum of MSC (GIBCO, AUS), 1%GlutaMAX-I (Invitrogen), 1% penicillium mould-Streptomycin sulphate liquid (Invitrogen), 10 μ M tynofovirs (LGM pharmaceuticals), concentration be the FGF-b (Invitrogen) of 20ng/ml; The medium component that in Comparative Examples 8, uses is: the DMEM of 88% (volume percent) (invitrogen) (this is basic medium), 1% L-glutaminate liquid (invitrogen), 1% penicillium mould-Streptomycin sulphate liquid (invitrogen), 10 μ M tynofovirs (LGM pharmaceuticals).
The inoculation quantity that cell goes down to posterity for the first time is 1 * 10
5, the density of cell inoculation is 1000/cm
2
Cell amplification quantity among result: the embodiment 6 is obviously greater than the cell amplification quantity in the Comparative Examples 8.Specifically referring to the broken line graph among Fig. 6.
Mixture through in the substratum that goes down to posterity of the mesenchymal stem cells MSCs that infects adult crab eating monkeys, adding antiretroviral drugs such as tynofovir, FGF-b or FGF-b and EGF etc.; And through with the medium component that cell amplification quantity and speed are increased among the present invention, make the first-generation 1 * 10
5Individual cell can increase 6～8 * 10
7Make the mesenchymal stem cells MSCs amplification of adult crab eating monkeys can satisfy the needs of test pair cell quantity, have great importance for the development of experiment before various therapies or medicine clinical.
In this experiment; We adopt with the go down to posterity mesenchymal stem cells MSCs of the adult crab eating monkeys cultivated of the condition among the embodiment 6 in embodiment 7; Promptly the special-purpose foetal calf serum of the α MEM (Invitrogen) of 88% (volume percent), MSC (GIBCO, AUS), 1%GlutaMAX-I (Invitrogen), 1% penicillium mould-Streptomycin sulphate liquid (Invitrogen), 10 μ M tynofovirs (LGM pharmaceuticals), concentration be the FGF-b (Invitrogen) of 20ng/ml; When cell grew into for the 4th generation, change substratum into do not comprise tynofovir substratum, all the other compositions are identical with above-mentioned substratum.Carrying out with the slow virus after 6 days when growth in the substratum that does not comprise tynofovir is the virus transfection of carrier, detects cell after 20 hours by the efficient of virus transfection.In Comparative Examples 9, use with embodiment 7 in identical condition, just in the process of cell cultures and virus transfection, all use tynofovir always.Below be the process of lentivirus preparation and transfection:
The first step: the preparation of slow virus (promptly in the 293T cell, carrying out the amplification of virus)
1) goes down to posterity and cultivate the 293T cell, keep cell, passed by 1: 4～1: 5 minute in higher density;
2) in transfection virus previous day, encapsulate 100mm petridish (making more jail of cell attachment) with poly-dextrorotation-lysine solution of 100 μ g/ml, room temperature effect 60min washes twice with aseptic PBS before the use;
3) make the 293T cell before transfection, can reach 70～80% remittance sheets;
4) get 400 μ l OPTI-MEM substratum (Invitrogen) and place the 5ml polystyrene tube; With vector plasmid DNA according to DUET101: CMV8.91: PMD.G=3: ratio was dissolved in wherein in 4: 1, are are these three kinds of vector plasmid DNA faced from John Hopkins University's journey and encouraged laboratory (http://www.addgene.org/pgvec1? F=c&identifier=17629&cmd=findpl&attag=c).Three DNA total amounts are 16 μ g, mixing, and room temperature is placed 5min;
5) another fills in the polystyrene tube of 400 μ l OPTI-MEM (Invitrogen) to get Lipofectamine2000 liposome (GIBCO) 32 μ l adding equally, soft mixing, and room temperature is placed 5min;
6) again with step 4) and 5) two kinds of liquid mixing obtaining, room temperature is placed 20min;
7) the mixed solution adding with DNA and Lipofectamine2000 liposome contains in the 293T Tissue Culture Dish of 8ml fresh culture mixing, 37 ℃, 5%CO
2Cultivate in the incubator;
8) 12h after the transfection discards transfection liquid, adds 8ml ITS nutrient solution and collects viral supernatant;
9) continue to collect viral supernatant behind cultivation 24hr, the 48hr, the centrifugal removal cell debris of 500rpm is filled in the aseptic centrifuge tube with 0.45 μ m filter, before concentrating, under 4 ℃, preserves.
Second step: the concentrating of slow virus
1) gets the aseptic DPBS of 4ml and be added on Ultra-Plus 20 filter membranes (Millipore), centrifugal 5 minutes of 2500rpm, moistening filter membrane.Discard pipe end DPBS solution.
2) will add in the filter membrane sleeve pipe through the filtering viral supernatant of 0.45 μ m filter, 4 ℃, the centrifugal 20min of 4000g.
3) supernatant on the absorption filter membrane plug-in unit is sub-packed in the 1.5ml EP pipe, and titre is stored in-80 ℃ of refrigerators after identifying, and is subsequent use.
The 3rd step: the evaluation of slow virus titre
1) poly-dextrorotation-lysine solution with 100 μ g/ml encapsulates six orifice plates, with every hole 2 * 10
5The density of individual cell with the 293T cell inoculation in six orifice plates.
2) viral supernatant is added in the 293T cell culture fluid, with the concentration adding polybrene of 8 μ g/ml.
3) fluorescent microscope calculates the green fluorescent protein positive cell number down, calculates viral supernatant titre with following formula.The volume of virus titer=green fluorescent protein positive cell percentage * 293 TCSs/viral supernatant of adding.
The 4th step: slow-virus transfection cell
1) according to green fluorescent protein (GFP) virus titer and cell quantity, equal 10 according to the MOI value and add viral supernatant (MOI=virus number/cell count promptly is equivalent to cell of 10 viruses), add polybrene with the concentration of 10 μ g/ml.Hatch after 20 hours and discard transfection liquid, add fresh culture;
2) after 1 week of cultivation, fluorescent microscope is observed down, calculates transfection efficiency in different time points simultaneously.
The result: slow-virus transfection efficient can reach 55% in embodiment 7, and the slow-virus transfection efficient in the Comparative Examples 9 has only about 10%.It is thus clear that utilize method of the present invention can improve the transfection efficiency of adult crab eating monkeys MSC cell widely.Can be referring to Fig. 7 and Fig. 8.
The above is merely the preferred embodiments of the present invention, is not limited to the present invention, and for a person skilled in the art, the present invention can have various changes and variation.All within spirit of the present invention and principle, any modification of being done, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (6)
1. the method for a vitro culture bone marrow mesenchymal stem cells of adult crab eating monkeys comprises:
I) to the said mesenchymal stem cells MSCs from said adult crab eating monkeys carry out former be commissioned to train foster;
II) when the degree of converging of said growth of marrow mesenchyme stem cell to 70%～80%, digest, go down to posterity and cultivate;
III) when the said cultured cells that goes down to posterity grows into 70%～80% degree of converging, digest once more, go down to posterity and cultivate;
What the said cultivation of going down to posterity was used is basic medium, in said basic medium, is added with foetal calf serum, penicillium mould and Streptomycin sulphate;
It is characterized in that: in the said basic medium of cultivating that is added with foetal calf serum, penicillium mould and Streptomycin sulphate that goes down to posterity, be added with antiretroviral drugs.
2. method according to claim 1 is characterized in that: said antiretroviral drugs is a tynofovir.
3. method according to claim 1 is characterized in that: the mixture that in said basic medium, adds FGF-b or FGF-b and EGF.
4. method according to claim 3 is characterized in that: said FGF-b or said EGF are respectively recombinant human FGF-b or recombinant human EGF.
5. according to claim 1 or 4 described methods, it is characterized in that: also be added with the GlutaMAX-I dipeptides in the said basic medium, said basic medium is a α lpha modified version MEM substratum.
6. method according to claim 5 is characterized in that: said foetal calf serum is the special-purpose foetal calf serum of mesenchymal stem cells MSCs.
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| CN1439717A (en) * | 2003-03-28 | 2003-09-03 | 浙江大学 | Method for separating and in vitro culturing stem cells |
| CN101412987A (en) * | 2008-03-04 | 2009-04-22 | 中国人民解放军第三军医大学第一附属医院 | Method for amplifying in vitro mesenchymal stem cells |
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| CN1439717A (en) * | 2003-03-28 | 2003-09-03 | 浙江大学 | Method for separating and in vitro culturing stem cells |
| CN101412987A (en) * | 2008-03-04 | 2009-04-22 | 中国人民解放军第三军医大学第一附属医院 | Method for amplifying in vitro mesenchymal stem cells |
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