CN104480068A - Method of in-vitro amplification and purification culture of mesenchymal stem cells - Google Patents
Method of in-vitro amplification and purification culture of mesenchymal stem cells Download PDFInfo
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Abstract
The invention relates to a method of in-vitro amplification and purification culture of mesenchymal stem cells in differentiation from mesenchymal stem cells to osteoblasts. A used cell culture medium comprises the following components: 5-20% of PL platelet lysate, 2-5U/ml of heparin, 5-10microgram/ml of doxycycline, and a basic culture medium DMEM or a basic culture medium AMEM. According to the method disclosed by the invention, the method comprising steps of separation and extraction of mesenchymal stem cells, cell inoculation, cell culture, cell culture fluid replacement and cell propagation are adopted, and the obtained mesenchymal stem cells are high in purity, high in in-vitro amplification capacity, and good in the capacity of differentiation to osteoblasts.
Description
Technical field
The present invention relates to the collection of mesenchymal stem cells MSCs, separation and Extraction, cell cultivates the technical field of amplification purification in vitro, specifically a kind of mesenchymal stem cells MSCs in vitro amplification purification cultivate method.
Background technology
Mesenchymal stem cells MSCs derives from grows early stage mesoderm, it is non-terminally differentiated cells, it has the ability of multinomial differentiation, correlative study has shown that this stem cell can be divided into skeletonization, cartilage, fat, and neurocyte etc., this cell is easy to gather, separation and Culture, and there is very strong self in vitro, the advantage such as differentiation capability and immunosuppression, make it in histoorgan defect disease, histoorgan degenerative disease and cell therapy and field of tissue engineering technology have important application prospect, there are in-vitro separation and the cultural method of numerous research and probe mesenchymal stem cells MSCs in recent years at home and abroad, mainly contain cell attachment to be separated, the method such as density gradient centrifugation and cell sorting, result display is low by the cell purity of adherent separation of going down to posterity, gradient centrifugation and cell sorting can obtain the cell of higher degree, but cell viability is relatively low, and required marrow amount is larger, affect follow-up investigation and application, the mesenchymal stem cells MSCs simultaneously obtained by these methods will lose the ability of amplification gradually in the process of Secondary Culture.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, provide a kind of mesenchymal stem cells MSCs in vitro amplification purification cultivate method.
The technical solution used in the present invention is: a kind of mesenchymal stem cells MSCs in vitro amplification purification cultivate method, it is characterized in that, described method comprises:
(1) collection of marrow blood: from posterior superior iliac spine bone marrow extraction blood.
(2) separation and Extraction of karyocyte:
A, density gradient centrifugation: injected by marrow blood and carry out centrifugal in sterile centrifugation tube a, centrifugal rear marrow blood is divided into three layers in centrifuge tube, and upper strata is supernatant liquor, and middle layer is the karyocyte layer of the cotton-shaped tunica albuginea layer of thin layer, and lower floor is red blood cell layer;
The separation and Extraction of B, karyocyte layer: first time karyocyte layer separation and Extraction: the karyocyte layer getting the supernatant liquor on upper strata and the tunica albuginea layer in middle layer is put into sterile centrifugation tube b and is carried out centrifugal, centrifugal rear taking-up upper strata supernatant liquor put back to carry out in centrifuge tube a centrifugal; The separation and Extraction of second time karyocyte layer: the karyocyte layer getting the supernatant liquor on upper strata and the tunica albuginea layer in middle layer is put into sterile centrifugation tube b and carried out centrifugal.
(3) cell counting: mixed by the karyocyte layer of extracted twice with PBS, gets 100 μ l cell suspensions and counts karyocyte and red corpuscle.
(4) cell inoculation: be 0.8-1.0 × 10 by initial density respectively according to the karyocyte of counting and red corpuscle
6/ cm
2with 1.0-1.2 × 10
8/ cm
2be inoculated in Tissue Culture Flask.
(5) cultivation of primary cell: the karyocyte of separation and Extraction is mixed with the perfect medium being suitable for primary cell culture, be inoculated in Tissue Culture Flask, cultivate in the cell culture incubator again Tissue Culture Flask filling cell being placed in saturated humidity, CO in cell culture incubator
2be 5%, O
2be 5%, temperature is 37 DEG C, when incubation time is 24-72h; Carry out cell and change liquid, discard the nutrient solution in culturing bottle, wash 2 Tissue Culture Flask cell attachment faces with PBS, after washing, add the perfect medium that the primary cell culture of new configuration is used; Every the perfect medium that replacing in three days primary cell culture is used, when cell grows to more than 80%, collection is carried out to cell and goes down to posterity.
(6) primary cell is collected and is gone down to posterity: a, the substratum discarded in culturing bottle, washs culturing bottle cell attachment face 2 times, discard PBS with PBS; B, in culturing bottle, add 0.25% pancreatin, be less than or equal to 3 minutes in room temperature digestion; C, when cell comes off from Tissue Culture Flask adherent beginning, add the perfect medium that cell cultures after the first-generation and the first-generation is used in Tissue Culture Flask, stop digestion; D, get 100 μ l cell suspensions for cell counting, by centrifugal extraction cell, cell-seeding-density is 6000-10000/cm
2cultivate.The number of days that the speed that the height of cell-seeding-density wants visual cell to increase and estimating is cultivated and determining.
(7) first-generation and the later cell cultures of the first-generation and go down to posterity: a, the Tissue Culture Flask filling cell is placed in saturated humidity cell culture incubator in cultivate, CO in cell culture incubator
2be 5%, temperature is 37 DEG C; B, when cell grows to more than 80%; C, the substratum discarded in culturing bottle, wash culturing bottle cell attachment face 2 times with PBS, discard PBS; D, in culturing bottle, add 0.25% pancreatin, be less than or equal to 3 minutes in room temperature digestion; E, when cell comes off from Tissue Culture Flask adherent beginning, add the perfect medium that cell cultures after the first-generation and the first-generation is used in Tissue Culture Flask, stop digestion; F, get 100 μ l cell suspensions for cell counting, by centrifugal extraction cell, cell-seeding-density is 6000-10000/cm
2cultivate.
The perfect medium that cell cultures is used:
The final concentration of the perfect medium that primary cell culture is used is composed as follows: PL platelet lysates liquid 5-20%, and heparin 2-5U/ml, doxycycline 5-10 μ g/ml, basic medium is DMEM.
After the first-generation and the first-generation, the final concentration of the perfect medium that cell cultures is used is composed as follows: PL platelet lysates liquid 5-20%, heparin 2-5U/ml, doxycycline 5-10 μ g/ml, basic medium is AMEM.
0.25% described pancreatin contains 0.02%EDTA.
The invention has the beneficial effects as follows:
1. the marrow blood of human body posterior superior iliac spine is abundanter, is easy to extract out and can obtain enough mesenchymal stem cells MSCs.
2. utilize density gradient centrifugation merely when medulla mesenchyma cellular segregation is extracted, do not add the reagent such as cellular segregation liquid, decrease the injury of extraneous agents on cellular, decrease the cost of experiment simultaneously, and a large amount of mescenchymal stem cells can be obtained.
3. different according to cell attachment speed, change cell culture fluid in early days, remove the hemopoietic stem cell in marrow, the cell debris existed in the cell of other types and supernatant liquor, is beneficial to the earlier purification of cell.
Accompanying drawing explanation
After Fig. 1 changes complete culture solution, the original cuiture cellular form of the 2nd day.
The 8th day of Fig. 2 culturing cell, population of cells slowly merge to more than 80% time cellular form.
The 11st day of Fig. 3 culturing cell is now first-generation cell, cellular form when growing to more than 80%.
The 14th day of Fig. 4 culturing cell is now s-generation cell, cellular form when growing to more than 80%.
The 17th day of Fig. 5 culturing cell is now s-generation cell, cellular form when growing to more than 80%.
Embodiment:
Below in conjunction with specific embodiment, the present invention is described further:
Embodiment 1:
Mesenchymal stem cells MSCs in vitro amplification purification cultivate a method, comprise the following steps:
(1) collection of marrow blood: extract 60ml marrow blood from the posterior superior iliac spine of volunteer.
(2) separation and Extraction of karyocyte:
A, density gradient centrifugation: injected by marrow blood and carry out centrifugal in sterile centrifugation tube a, centrifugal rear marrow blood is divided into three layers in centrifuge tube, and upper strata is supernatant liquor, and middle layer is the karyocyte layer of the cotton-shaped tunica albuginea layer of thin layer, and lower floor is red blood cell layer; Density gradient centrifugation is carried out when not adding any cellular segregation liquid.The separation and Extraction of B, karyocyte layer: first time karyocyte layer separation and Extraction: the karyocyte layer getting the supernatant liquor on upper strata and the tunica albuginea layer in middle layer is put into sterile centrifugation tube b and is carried out centrifugal, centrifugal rear taking-up upper strata supernatant liquor put back to carry out in centrifuge tube a centrifugal; When tunica albuginea layer and supernatant liquor are taken out, in order to obtain more karyocyte, a small amount of red corpuscle can be carried secretly, put into sterile centrifugation tube b, tunica albuginea layer will be filled, supernatant liquor and a small amount of erythrocytic centrifuge tube carry out centrifugal, and centrifugal rear taking-up can not be put back in centrifuge tube a with tunica albuginea layer and erythrocytic supernatant liquor.The separation and Extraction of second time karyocyte layer: the karyocyte layer getting the supernatant liquor on upper strata and the tunica albuginea layer in middle layer is put into sterile centrifugation tube b and carried out centrifugal.Step is with the separation and Extraction of first time karyocyte layer.
(3) cell counting: mixed by the karyocyte layer of extracted twice with PBS, gets 100 μ l cell suspensions and counts karyocyte and red corpuscle;
(4) cell inoculation: karyocyte and red corpuscle are counted, the area of required Tissue Culture Flask is calculated according to its quantity, then according to the volume of the required perfect medium of the quantity of Tissue Culture Flask preparation, be 0.8-1.0 × 10 by initial density respectively by the complete karyocyte of counting and red corpuscle
6/ cm
2with 1.0-1.2 × 10
8/ cm
2be inoculated in Tissue Culture Flask.
(5) cultivation of primary cell: now with the perfect medium of primary cell culture, cell is mixed, be inoculated in Tissue Culture Flask, then cultivate in the cell culture incubator Tissue Culture Flask filling cell being placed in saturated humidity, CO in cell culture incubator
2be 5%, O
2be 5%, temperature is 37 DEG C, when incubation time is 24-72h; Carry out cell and change liquid, discard the nutrient solution in culturing bottle, wash 2 Tissue Culture Flask cell attachment faces with PBS, after washing, add the perfect medium that the primary cell culture of new configuration is used; Every the complete cell culture medium that replacing in three days primary cell culture is used, when cell grows to more than 80%, collection is carried out to cell and goes down to posterity.
(6) primary cell is collected and is gone down to posterity: discard the substratum in culturing bottle, washs culturing bottle cell attachment face 2 times with PBS, and in the process of washing, during double swerve culturing bottle, action is wanted slowly, to avoid cell to be washed from cell attachment face by PBS, discard PBS; In culturing bottle, add 0.25% pancreatin, 0.25% described pancreatin contains 0.02%EDTA, is no more than 3 minutes in room temperature digestion; When cell comes off from Tissue Culture Flask adherent beginning, now pat the sidewall of Tissue Culture Flask, after cell all falls from the cell attachment emaciated face of Tissue Culture Flask, the perfect medium of cell cultures after the first-generation and the first-generation is added in culturing bottle, stops peptic cell; Get 100 μ l cell suspensions for cell counting, by centrifugal extraction cell, cell-seeding-density is 6000-10000/cm
2cultivate.
(7) first-generation and the later cell cultures of the first-generation and go down to posterity: a, the Tissue Culture Flask filling cell is placed in saturated humidity cell culture incubator in cultivate, CO in cell culture incubator
2be 5%, temperature is 37 DEG C; B, when cell grows to more than 80%; C, the substratum discarded in culturing bottle, wash culturing bottle cell attachment face 2 times with PBS, discard PBS; D, in culturing bottle, add 0.25% pancreatin, be less than or equal to 3 minutes in room temperature digestion; E, when cell comes off from Tissue Culture Flask adherent beginning, add the perfect medium that cell cultures after the first-generation and the first-generation is used in Tissue Culture Flask, stop digestion; F, get 100 μ l cell suspensions for cell counting, by centrifugal extraction cell, cell-seeding-density is 6000-10000/cm
2cultivate.
Above-mentioned mesenchymal stem cells MSCs in vitro amplification purification cultivates the preparation of all perfect mediums:
The final concentration of the perfect medium that primary cell culture is used is composed as follows: PL platelet lysates liquid 5-20%, and heparin 2-5U/ml, doxycycline 5-10 μ g/ml, basic medium is DMEM nutrient solution.
The final concentration of the first-generation and the later cell cultures perfect medium used of the first-generation is composed as follows: PL platelet lysates liquid 5-20%, heparin 2-5U/ml, doxycycline 5-10 μ g/ml, basic medium is AMEM nutrient solution.
Embodiment 2:
1. the collection of marrow blood: extract 60ml marrow blood from the posterior superior iliac spine of volunteer
2. the separation and Extraction of karyocyte (mainly mesenchymal stem cells MSCs): the marrow blood equivalent of 60ml is put into the centrifuge tube of numbering and being respectively 1. and 2., at 200g, density gradient centrifugation is carried out under the condition of 6min room temperature, centrifugal rear marrow blood is divided into three layers, upper strata is supernatant liquor, middle layer is the karyocyte layer of very thin white flock, lower floor is red blood cell layer, now carry out the separation and Extraction of first time karyocyte layer: will to be numbered 1. and karyocyte layer 2. in centrifuge tube and supernatant liquor take out respectively, in order to obtain more karyocyte, some red corpuscle can be got less, put into the sterile centrifugation tube be numbered 3., by the centrifuge tube that is numbered 3. at 1000g, the condition of 6min room temperature is carried out centrifugal, in the centrifuge tube that centrifugal rear taking-up can not be divided into equivalent two parts to put back to being numbered 1. and 2. with tunica albuginea layer and erythrocytic supernatant liquor, again the separation and Extraction of second time karyocyte is carried out after mixing, step is with the separation and Extraction of first time karyocyte.
3. cell counting: mixed by the karyocyte of extracted twice with PBS, is placed in the centrifuge tube that is numbered 5., gets 100 μ l cell suspensions and count karyocyte and red corpuscle, count results: karyocyte 451.5 × 10
6, red corpuscle 572.25 × 10
8.
4. cell inoculation: according to karyocyte and red blood cell count(RBC), calculate the area with Tissue Culture Flask, again according to the volume of the quantity of Tissue Culture Flask preparation primary cell culture perfect medium used, be 0.86 × 10 by initial density respectively by the complete karyocyte of counting and red corpuscle
6/ cm
2with 1.09 × 10
8/ cm
2be inoculated in 3 175cm
2in Tissue Culture Flask.
5. the cultivation of primary cell: the Tissue Culture Flask filling cell is placed in CO
2be 5%, O
2be 5%, temperature is 37 DEG C, cultivate in the cell culture incubator of saturated humidity, cultivate by 48 hours, cell carries out changing liquid, discard the nutrient solution in culturing bottle, with PBS washed cell culturing bottle cell attachment face 2 times gently, the perfect medium that primary cell culture is used is added after discarding PBS, the cell picture of 48 hours is shown in (Fig. 1), cell is long shuttle-type, group's formula growth, the complete cell culture medium that primary cell culture is used is changed at the 5th day, liquid is changed with first time, when within the 8th day, cell grows to more than 80%, cell picture is shown in (Fig. 2), cell is long shuttle-type, vartex grows, cell is carried out collection go down to posterity.
6. primary cell is collected and is gone down to posterity: discard the substratum in culturing bottle, culturing bottle cell attachment face is washed gently 2 times with PBS, discard PBS, each Tissue Culture Flask adds 0.25% pancreatin 6ml, pancreatin contains 0.02%EDTA, room temperature digestion is no more than 3 minutes, when cell falls from the adherent emaciated face of Tissue Culture Flask, add all perfect mediums of 6ml first-generation cell cultures in each Tissue Culture Flask and stop digestion, all cell suspensions are put into a container to mix, get 100 μ l cell suspensions for cell counting, learn that the counting total amount of mesenchymal stem cells MSCs is 15.2 × 10 by cell counting
6, be inoculated in by cell in 11 T175 Tissue Culture Flasks, cell-seeding-density is 7896/cm
2cultivate.
7. the cultivation amplification purification of first-generation cell and later generation cell: the Tissue Culture Flask filling cell is placed in CO
2be 5%, temperature is 37 DEG C, cultivate in the cell culture incubator of saturated humidity, every day observation of cell form and degrees of fusion, at the 11st day of cell cultures, cell grows to more than 80%, cell picture is shown in (Fig. 3), now by passage, continue to cultivate amplification purification, it is identical that step and the primary cell of passage collect the step gone down to posterity, at the 14th day of cell cultures, cell grows to more than 80%, see (Fig. 4), now continue passage amplification purification, at the 17th day of cell cultures, cell grows to more than 80%, cell picture is shown in (Fig. 5).
Mesenchymal stem cells MSCs in vitro amplification purification cultivates the preparation of perfect medium used:
The preparation of primary cell culture perfect medium used: perfect medium 90ml needed for Tissue Culture Flask, final concentration is composed as follows, PL platelet lysates liquid adds by 10%, needs 9ml, and heparin is pressed 5U/ml and added, need 450U, doxycycline adds by 8 μ g/ml, needs 720 μ g, needs basic medium DMEM 81ml, 37 DEG C of thermostat water bath preheating 5min are placed on after having prepared, stand-by.
The perfect medium preparation that first-generation cell cultures is used: perfect medium 330ml needed for Tissue Culture Flask, final concentration is composed as follows, PL platelet lysates liquid adds by 10%, needs 33ml, and heparin is pressed 5U/ml and added, need 1650U, doxycycline adds by 8 μ g/ml, needs 2640 μ g, needs basic medium AMEM207ml, 37 DEG C of thermostat water bath preheating 5min are placed on after having prepared, stand-by.
Due to mesenchymal stem cells MSCs adherent growth, the characteristics such as the adherent time is very fast, constantly purifying is obtained in the process of cell cultures, simultaneously in order to prevent cell from the process of cultivating, cell senescence occurring, variation, the danger that differentiation etc. are potential, general cell cultures number of days is no more than 21 days, and cell cultures generation is at P3 or P4.The detection of mesenchymal stem cells MSCs surface antigen: get the attached cell that the 3rd generation grew to more than 80%, with tryptic digestion, get 2 × 10 after counting
6individual cell, packing 6 is managed, and often pipe adds 10 μ l fluorescent-labeled antibody CD45-FITC, CD34-PE, CD73-PE, CD105-FITC, CD166-PE, separately sets 1 pipe as blank, room temperature lucifuge 30 minutes; Repeatedly wash 2-3 time with PBS, reduce non-specific binding; Add 200 μ lPBS and mix cell, and fix with 1% paraformaldehyde, 4 DEG C of preservations, in 24 hours, flow cytometer detects, learn through flow cytometer phenotype analytical, express mesenchymal stem cells MSCs mark CD73 (98.6%), CD105 (97.7%), CD166 (97.4%), do not express hematopoietic cell markers CD45 (0.2%), CD34 (0.4%), this detection shows that the purity of mesenchymal stem cells MSCs is higher.
Claims (4)
1. mesenchymal stem cells MSCs in vitro amplification purification cultivate a method, it is characterized in that, described method comprises:
(1) collection of marrow blood: from posterior superior iliac spine bone marrow extraction blood.
(2) separation and Extraction of karyocyte:
A, density gradient centrifugation: injected by marrow blood and carry out centrifugal in sterile centrifugation tube a, centrifugal rear marrow blood is divided into three layers in centrifuge tube, and upper strata is supernatant liquor, and middle layer is the karyocyte layer of the cotton-shaped tunica albuginea layer of thin layer, and lower floor is red blood cell layer;
The separation and Extraction of B, karyocyte layer: first time karyocyte layer separation and Extraction: the karyocyte layer getting the supernatant liquor on upper strata and the tunica albuginea layer in middle layer is put into sterile centrifugation tube b and is carried out centrifugal, centrifugal rear taking-up upper strata supernatant liquor put back to carry out in centrifuge tube a centrifugal; The separation and Extraction of second time karyocyte layer: the karyocyte layer getting the supernatant liquor on upper strata and the tunica albuginea layer in middle layer is put into sterile centrifugation tube b and carried out centrifugal.
(3) cell counting: mixed by the karyocyte layer of extracted twice with PBS, gets 100 μ l cell suspensions and counts karyocyte and red corpuscle.
(4) cell inoculation: be 0.8-1.0 × 10 by initial density respectively according to the karyocyte of counting and red corpuscle
6/ cm
2with 1.0-1.2 × 10
8/ cm
2be inoculated in Tissue Culture Flask.
(5) cultivation of primary cell: the cell mixing of will extract with the perfect medium that primary cell culture is used, be inoculated in Tissue Culture Flask, cultivate in the cell culture incubator Tissue Culture Flask filling cell being placed in saturated humidity, CO in cell culture incubator
2be 5%, O
2be 5%, temperature is 37 DEG C, when incubation time is 24-72h; Carry out cell and change liquid, discard the nutrient solution in culturing bottle, wash 2 Tissue Culture Flask cell attachment faces with PBS, after washing, add the perfect medium that the primary cell culture of new configuration is used; Every the perfect medium that replacing in three days primary cell culture is used, when cell grows to more than 80%, collection is carried out to cell and goes down to posterity.
(6) primary cell is collected and is gone down to posterity: a, the substratum discarded in culturing bottle, washs culturing bottle cell attachment face 2 times, discard PBS with PBS; B, in culturing bottle, add 0.25% pancreatin, be less than or equal to 3 minutes in room temperature digestion; C, when cell comes off from Tissue Culture Flask adherent beginning, the perfect medium adding cell cultures after the first-generation and the first-generation used adds in culturing bottle, stops digestion; D, get 100 μ l cell suspensions for cell counting, by centrifugal extraction cell, cell-seeding-density is 6000-10000/cm
2cultivate.
(7) first-generation and the later cell cultures of the first-generation and go down to posterity: a, the Tissue Culture Flask filling cell is placed in saturated humidity cell culture incubator in cultivate, CO in cell culture incubator
2be 5%, temperature is 37 DEG C; B, when cell grows to more than 80%; C, the substratum discarded in culturing bottle, wash culturing bottle cell attachment face 2 times with PBS, discard PBS; D, in culturing bottle, add 0.25% pancreatin, be less than or equal to 3 minutes in room temperature digestion; E, when cell comes off from Tissue Culture Flask adherent beginning, add the perfect medium that cell cultures after the first-generation and the first-generation is used in Tissue Culture Flask, stop digestion; F, get 100 μ l cell suspensions for cell counting, by centrifugal extraction cell, cell-seeding-density is 6000-10000/cm
2cultivate.
2. mesenchymal stem cells MSCs according to claim 1 in vitro amplification purification cultivate method, it is characterized in that the preparation of the perfect medium that described primary cell culture is used: final concentration is composed as follows, PL platelet lysates liquid 5-20%, heparin 2-5U/ml, doxycycline 5-10 μ g/ml, basic medium is DMEM nutrient solution.
3. mesenchymal stem cells MSCs according to claim 1 in vitro amplification purification cultivate method, it is characterized in that the preparation of the perfect medium that cell cultures is used after the described first-generation and the first-generation: final concentration is composed as follows, PL platelet lysates liquid 5-20%, heparin 2-5U/ml, doxycycline 5-10 μ g/ml, basic medium is AMEM nutrient solution.
4. mesenchymal stem cells MSCs according to claim 1 in vitro amplification purification cultivate method, it is characterized in that 0.25% described pancreatin contains 0.02%EDTA.
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Application publication date: 20150401 |