CN104450613A - Cell culture medium beneficial to in-vitro culture of autologous bone marrow mesenchymal stem cells - Google Patents
Cell culture medium beneficial to in-vitro culture of autologous bone marrow mesenchymal stem cells Download PDFInfo
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- CN104450613A CN104450613A CN201410776851.5A CN201410776851A CN104450613A CN 104450613 A CN104450613 A CN 104450613A CN 201410776851 A CN201410776851 A CN 201410776851A CN 104450613 A CN104450613 A CN 104450613A
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Abstract
The invention relates to the technical field of cell culture media, and discloses a cell culture medium beneficial to in-vitro culture of autologous bone marrow mesenchymal stem cells. The cell culture medium is characterized in that an autologous PL blood platelet lysis buffer is added during preparation. The autologous bone marrow mesenchymal stem cells are cultured by using the cell culture medium added with the PL blood platelet lysis buffer so as to ensure that the cell proliferation capability of cells in in-vitro culture; and meanwhile, a phenomenon that the mesenchymal stem cells cannot be affected by exogenous pollution in in-vitro culture and amplification can be ensured, the immunological rejection of allogeneic cells generated in a disease treatment process can also be avoided, and an application of treating self-diseases by using autologous cells clinically can be facilitated.
Description
Technical field
The present invention relates to the technical field of cell culture medium, be specifically a kind ofly beneficial to the cell culture medium that autologous bone marrow mesenchymal stem cells cultivates amplification in vitro.The cell culture medium of autologous PL platelet lysates liquid is added in cell culture medium.
Background technology
In the last few years, mesenchymal stem cells MSCs is cultivating cell culture medium used in the process of amplification, generally all will add foetal calf serum, what have also will add some other cell growth factor, maintains the cultivation amplification of cell, when adding foetal calf serum in cell culture medium, can by foetal calf serum containing foreign protein matter, also likely Carried bacteria, virus, protein communicable disease or Protein virus etc. are mixed into cell, and these cells will be unfavorable for treatment use clinically.The present invention substitutes foetal calf serum and some other cell growth factor with autologous PL platelet lysates liquid in the preparation of cell culture medium, autologous mesenchymal stem cells MSCs is cultivated with the cell culture medium adding PL platelet lysates liquid, the multiplication capacity of cell when can ensure that cell is cultivated in vitro, also ensure that mesenchymal stem cells MSCs is not subject to ectogenic pollution when cultivating amplification in vitro simultaneously, also avoid immunological rejection during variant cell disease therapy, be conducive to the application using autologous cell therapy self-disease clinically.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, provide a kind of and be beneficial to the cell culture medium that autologous bone marrow mesenchymal stem cells cultivates amplification in vitro.
The technical solution used in the present invention is:
Be beneficial to the cell culture medium that autologous bone marrow mesenchymal stem cells cultivates amplification in vitro, it is characterized in that:
The perfect medium that described primary cell culture is used, its final concentration is composed as follows: PL platelet lysates liquid 5-20%, heparin 2-5U/ml, doxycycline 5-10 μ g/ml, substratum based on solvent, and basic medium is DMEM nutrient solution;
The described first-generation and the first-generation perfect medium that cell cultures is used later, its final concentration is composed as follows: PL platelet lysates liquid 5-20%, heparin 2-5U/ml, doxycycline 5-10 μ g/ml, substratum based on solvent, basic medium is AMEM nutrient solution.
Be beneficial to the preparation method that autologous bone marrow mesenchymal stem cells cultivates the cell culture medium of amplification in vitro, it is characterized in that: described preparation method comprises:
(1) preparation of PL platelet lysates liquid: extract peripheral blood, after density gradient centrifugation, take out and be rich in hematoblastic supernatant liquor on upper strata, mix in sterile chamber, put into-20 DEG C of frozen at least 1h of refrigerator in packing centrifuge tube, needed for cell cultures, the amount of PL is thawed in 37 DEG C of thermostat water baths, the time of thawing is less than or equal to 5min, under the condition of room temperature, 1700g, 6min are centrifugal, get the preparation of supernatant liquor for substratum;
(2) preparation of the perfect medium that primary cell culture is used: its final concentration consists of PL platelet lysates liquid 5-20%, heparin 2-5U/ml, doxycycline 5-10 μ g/ml, substratum DMEM nutrient solution based on solvent;
(3) first-generation and the first-generation perfect medium preparation that cell cultures is used later: its final concentration is composed as follows, PL platelet lysates liquid 5-20%, heparin 2-5U/ml, doxycycline 5-10 μ g/ml, culture medium A MEM nutrient solution based on solvent.
Effect of the present invention:
1. the PL platelet lysates liquid preparing process in perfect medium is simple, extracts the comparatively small amt of peripheral blood, does not have detrimentally affect to human body.
2. cell culture medium can ensure the multiplication capacity of cell, obtains a large amount of mesenchymal stem cells MSCs at short notice.In the process of cell cultures, use the complete cell culture medium that two kinds are beneficial to autologous bone marrow mesenchymal stem cells amplification purification, be conducive to the amplification of cell.
3. cultivate autologous mesenchymal stem cells MSCs with autologous PL platelet lysates liquid culture medium, ensure that mesenchymal stem cells MSCs does not add external source serum in culturing process in vitro, it also avoid exogenous infection disease simultaneously, be conducive to mesenchymal stem cells MSCs treatment use clinically.
Accompanying drawing explanation
Fig. 1 peripheral blood is dispensed in 50ml centrifuge tube.
After density gradient centrifugation, there is the peripheral blood of layering in Fig. 2.
After Fig. 3 changes complete culture solution, the original cuiture cellular form of the 2nd day.
The 8th day of Fig. 4 culturing cell, population of cells slowly merge to more than 80% time cellular form.
The 11st day of Fig. 5 culturing cell is now first-generation cell, cellular form when growing to more than 80%.
The 14th day of Fig. 6 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:
A kind ofly be beneficial to the cell culture medium that autologous bone marrow mesenchymal stem cells cultivates amplification in vitro:
The perfect medium that described primary cell culture is used, its final concentration is composed as follows: PL platelet lysates liquid 5-20%, heparin 2-5U/ml, doxycycline 5-10 μ g/ml, substratum based on solvent, and basic medium is DMEM nutrient solution;
The described first-generation and the first-generation perfect medium that cell cultures is used later, its final concentration is composed as follows: PL platelet lysates liquid 5-20%, heparin 2-5U/ml, doxycycline 5-10 μ g/ml, substratum based on solvent, basic medium is AMEM nutrient solution.
Wherein PL accounts for the per-cent of cell culture complete medium is add according to the age of people, and the interpolation PL per-cent being less than or equal to 45 years old is 5-10%, and the interpolation PL per-cent being greater than 45 years old is 10-20%.
Be beneficial to the preparation method that autologous bone marrow mesenchymal stem cells cultivates the cell culture medium of amplification in vitro, described in it, preparation method comprises:
(1) preparation of PL platelet lysates liquid: extract peripheral blood, after density gradient centrifugation, take out and be rich in hematoblastic supernatant liquor on upper strata, mix in sterile chamber, put into-20 DEG C of frozen at least 1h of refrigerator in packing centrifuge tube, needed for cell cultures, the amount of PL is thawed in 37 DEG C of thermostat water baths, and the time of thawing is less than or equal to 5min, under the condition of room temperature, carry out 6min centrifugal, get the preparation of supernatant liquor for substratum.Be that the single use blood taking bag that 200ml has added low molecular weight heparin sodium 5000U extracts 200ml peripheral blood at volunteer's elbow joint place with volume, be transferred in 50ml sterile centrifugation tube by blood taking bag after peripheral blood mixing, the amount of often propping up centrifuge tube packing is 40ml, see Fig. 1, to the centrifuge tube of peripheral blood be filled at 200g, density gradient centrifugation is carried out under the condition of 20min room temperature, there is layering in the peripheral blood after centrifugal, hematoblastic supernatant liquor is on upper strata, be rich in centrifuge tube that hematoblastic supernatant liquor accounts for greatly peripheral blood volume 43%, see Fig. 2, being rich in can not with the red corpuscle of lower floor time hematoblastic supernatant liquor takes out, supernatant liquor is put into same sterile chamber to mix, be dispensed into again in the centrifuge tube of 15ml, the amount of often propping up centrifuge tube packing is 5-10ml, put into-20 DEG C of refrigerators frozen, after frozen at least 1h, needed for cell cultures, the amount of PL is thawed in 37 DEG C of thermostat water baths, the time of thawing is no more than 5min, at 1700g after thawing, carry out centrifugal under the condition of 6min room temperature, get the preparation of supernatant liquor for substratum.
(2) preparation of the perfect medium that primary cell culture is used: its final concentration consists of PL platelet lysates liquid 5-20%, heparin 2-5U/ml, doxycycline 5-10 μ g/ml, substratum DMEM nutrient solution based on solvent.Quantity according to culturing bottle is learnt, need the complete cell culture medium of preparation 60ml, final concentration is composed as follows: PL platelet lysates liquid adds by 10%, need 6ml, heparin adds by 4U/ml, need 240U, doxycycline to add by 8 μ g/ml, need 480 μ g, need basic medium DMEM 54ml.
According to age and each composition interpolation scope of people, the cell culture medium of multiple final concentration can be prepared, as following three kinds: 1. final concentration is composed as follows: PL platelet lysates liquid adds by 5% needs 3ml, heparin adds by 2U/ml needs 120U, doxycycline adds needs 300 μ g by 5 μ g/ml, basic medium DMEM 54ml.
2. final concentration is composed as follows: PL platelet lysates liquid adds by 10% needs 6ml, and heparin adds by 4U/ml needs 240U, and doxycycline adds needs 480 μ g by 8 μ g/ml, basic medium DMEM 54ml.
3. final concentration is composed as follows: PL platelet lysates liquid adds by 20% needs 12ml, and heparin adds by 5U/ml needs 300U, and doxycycline adds needs 600 μ g by 10 μ g/ml, basic medium DMEM 54ml.
The preparation of the perfect medium that primary cell culture is used: learnt by counting cells quantity and need with 2 175 cm
2tissue Culture Flask, learn according to the quantity of culturing bottle and need the complete cell culture medium of preparation 60ml, composed as follows eventually: PL adds according to 10% of complete cell culture medium cumulative volume, need PL6ml, heparin adds according to 5U/ml, need heparin 5U/ml × 60ml=300U, doxycycline adds according to 8 μ g/ml, need 8 μ g/ml × 60ml=480 μ g, need the amount of basic medium DMEM to be 60ml-6ml=54ml, the volume of heparin and the shared cell culture medium completely of doxycycline can be ignored.The complete cell culture medium prepared before use will in 37 DEG C of thermostat water baths preheating 5-10min.
(3) first-generation and the first-generation perfect medium preparation that cell cultures is used later: its final concentration is composed as follows, PL platelet lysates liquid 5-20%, heparin 2-5U/ml, doxycycline 5-10 μ g/ml, culture medium A MEM nutrient solution based on solvent.Quantity according to culturing bottle is learnt, need the complete cell culture medium of preparation 210ml, final concentration is composed as follows, and PL platelet lysates liquid 21ml, heparin 1050U, doxycycline 1680 μ g, the amount of basic medium AMEM nutrient solution is 189ml.
As following three kinds: 1. final concentration is composed as follows, PL platelet lysates liquid adds by 5% needs 10.5ml, and heparin adds by 2U/ml needs 420U, and doxycycline adds needs 1050 μ g by 5 μ g/ml, basic medium AMEM 199.5ml.
2. final concentration is composed as follows: PL platelet lysates liquid adds by 10% needs 21ml, and heparin adds by 4U/ml needs 840U, and doxycycline adds needs 1680 μ g by 8 μ g/ml, basic medium AMEM 189ml.
3. final concentration is composed as follows: PL platelet lysates liquid adds by 20% needs 42ml, and heparin adds by 5U/ml needs 1050U, and doxycycline adds needs 2100 μ g by 10 μ g/ml, basic medium AMEM168ml.
The first-generation and the first-generation perfect medium preparation that cell cultures is used later: learn according to the quantity of culturing bottle and need the complete cell culture medium of preparation 210ml, composed as follows eventually: PL accounts for 10% of complete cell culture medium cumulative volume to be added, need PL21ml, heparin adds according to 5U/ml, need heparin 5U/ml × 210ml=1050U, doxycycline adds according to 8 μ g/ml, need 8 μ g/ml × 210ml=1680 μ g, the amount of basic medium DMEM is needed to be 210ml-21ml=189ml, the volume of heparin and the shared cell culture medium completely of doxycycline can be ignored.
Above-mentionedly be beneficial to the cell culture medium that autologous bone marrow mesenchymal stem cells cultivates amplification in vitro, the application method in the cultivation of cell:
(1) mesenchymal stem cells MSCs is extracted: posterior superior iliac spine bone marrow extraction blood, extracting the consubstantiality posterior superior iliac spine bone marrow extraction blood of peripheral blood, after density gradient centrifugation, separation and Extraction karyocyte; The Tissue Culture Flask that cell inoculation needs is determined after cell counting.Extract 60ml marrow blood at the posterior superior iliac spine of same volunteer, through density gradient centrifugation, separation and Extraction karyocyte (being mainly mesenchymal stem cells MSCs), learns through cell counting, cell inoculation needs 2 175cm
2tissue Culture Flask.
(2) cultivation of primary cell: the karyocyte mixing that primary cell culture perfect medium used will be separated, now with the perfect medium being suitable for primary cell culture, the karyocyte of separation and Extraction is mixed, be inoculated in Tissue Culture Flask, be inoculated in Tissue Culture Flask, the Tissue Culture Flask filling cell is positioned over CO
2be 5%, O
2be 5%, temperature is 37 DEG C, cultivates in the cell culture incubator of saturated humidity, when cultivating 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, the perfect medium adding primary cell culture used is shown in Fig. 3; Changed the perfect medium that primary cell culture is used every three days, when cell grows to more than 80%, collection is carried out to cell and goes down to posterity.
(3) primary cell is collected and is gone down to posterity: discard the substratum in culturing bottle, culturing bottle cell attachment face is washed 2 times with PBS, culturing bottle cell attachment face is washed 2 times with PBS, in the process of washing, during double swerve culturing bottle, action is wanted slowly, avoid cell to be washed from cell attachment face by PBS, discard PBS; In culturing bottle, add 0.25% pancreatin, room temperature digestion is no more than 3 minutes; When cell falls from the adherent emaciated face of Tissue Culture Flask, add the first-generation and the first-generation perfect medium that cell cultures is used later, stop digestion, through centrifugal extraction mesenchymal stem cells MSCs, continue to cultivate amplification purification.When cell cultures was by the 8th day, see that Fig. 4 cell goes down to posterity, discard the substratum in culturing bottle, wash culturing bottle cell attachment face gently 2 times with PBS, discard PBS, add 0.25% pancreatin, pancreatin contains 0.02%EDTA, and room temperature digestion is generally no more than 3 minutes, when cell falls from the adherent emaciated face of Tissue Culture Flask, add the perfect medium prepared and stop digestion, by cell suspension mixing sampling counting, the quantity of mesenchymal stem cells MSCs is 10.2 × 10
6, cell inoculation needs 7 175cm
2tissue Culture Flask.
(4) first-generation and the later cell cultures of the first-generation and go down to posterity: the mesenchymal stem cells MSCs mixing that will extract with the perfect medium that cell cultures after the first-generation and the first-generation is used, be inoculated in Tissue Culture Flask, then the Tissue Culture Flask filling cell is placed in CO
2be 5%, temperature is 37 DEG C, cultivates in the cell culture incubator of saturated humidity, and by passage when cell grows to more than 80%, it is identical that step and the primary cell of passage collect the step gone down to posterity.When the 11st day of culturing cell, see Fig. 5, counting mesenchymal stem cells MSCs quantity is 40.2 × 10
6, when the 14th day of cell cultures, see Fig. 6, counting mesenchymal stem cells MSCs quantity is 151 × 10
6.From cell picture cells grown shuttle-type, stereoscopic sensation is strong, illustrates that cell is good, can find out that cell still maintains stronger amplification ability from cell counting.
The complete cell culture medium that two kinds are beneficial to autologous bone marrow mesenchymal stem cells amplification purification is used in the process of cell cultures, a kind of cultivation for primary cell, its preparation final concentration is composed as follows: PL platelet lysates liquid accounts for the 5-20% of perfect medium volume, heparin 2-5U/ml, doxycycline 5-10 μ g/ml, basic medium is DMEM nutrient solution; The another kind of first-generation and the first-generation of being used for is with the cultivation of progeny cell, and its preparation final concentration is composed as follows: PL platelet lysates liquid accounts for the 5-20% of perfect medium volume, heparin 2-5U/ml, and doxycycline 5-10 μ g/ml, basic medium is AMEM nutrient solution.
Can use in cell cultivation process different size with air-permeable envelope Tissue Culture Flask, add in culturing bottle cell culture medium, PBS, pancreatin, for stopping the amount of the cell culture medium of peptic cell, as follows:
25cm
2cell accompany foster bottle add substratum, PBS, pancreatin, termination digestion amount be respectively: 5ml, 1ml, 1ml, 1ml.
75cm
2cell accompany foster bottle add substratum, PBS, pancreatin, termination digestion amount be respectively: 10ml, 2ml, 2ml, 2ml.
150cm
2cell accompany foster bottle add substratum, PBS, pancreatin, termination digestion amount be respectively: 20ml, 4ml, 4ml, 4ml.
175cm
2cell accompany foster bottle add substratum, PBS, pancreatin, termination digestion amount be respectively: 30ml, 6ml, 6ml, 6ml.
500cm
2cell accompany foster bottle add substratum, PBS, pancreatin, termination digestion amount be respectively: 75ml, 15ml, 15ml, 15ml.
Claims (2)
1. be beneficial to the cell culture medium that autologous bone marrow mesenchymal stem cells cultivates amplification in vitro, it is characterized in that:
The perfect medium that described primary cell culture is used, its final concentration is composed as follows: PL platelet lysates liquid 5-20%, heparin 2-5U/ml, doxycycline 5-10 μ g/ml, substratum based on solvent, and basic medium is DMEM nutrient solution;
The described first-generation and the first-generation perfect medium that cell cultures is used later, its final concentration is composed as follows: PL platelet lysates liquid 5-20%, heparin 2-5U/ml, doxycycline 5-10 μ g/ml, substratum based on solvent, basic medium is AMEM nutrient solution.
2. be beneficial to the preparation method that autologous bone marrow mesenchymal stem cells cultivates the cell culture medium of amplification in vitro, it is characterized in that: described preparation method comprises:
(1) preparation of PL platelet lysates liquid: extract peripheral blood, after density gradient centrifugation, take out and be rich in hematoblastic supernatant liquor on upper strata, mix in sterile chamber, put into-20 DEG C of frozen at least 1h of refrigerator in packing centrifuge tube, needed for cell cultures, the amount of PL is thawed in 37 DEG C of thermostat water baths, and the time of thawing is less than or equal to 5min, under the condition of room temperature, carry out 6min centrifugal, get the preparation of supernatant liquor for substratum;
(2) preparation of the perfect medium that primary cell culture is used, its final concentration consists of: PL platelet lysates liquid 5-20%, heparin 2-5U/ml, doxycycline 5-10 μ g/ml, substratum DMEM nutrient solution based on solvent;
(3) preparation of the perfect medium that cell cultures is used after the first-generation and the first-generation, its final concentration consists of: PL platelet lysates liquid 5-20%, heparin 2-5U/ml, doxycycline 5-10 μ g/ml, culture medium A MEM nutrient solution based on solvent.
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Cited By (6)
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| CN106434530A (en) * | 2016-11-16 | 2017-02-22 | 沈阳细胞治疗工程技术研发中心有限公司 | Endothelial cell culture solution |
| CN106566802A (en) * | 2016-08-31 | 2017-04-19 | 山东景源生物科技有限公司 | Preparation method for preparing culture kit of umbilical cord blood mesenchemal stem cells |
| CN108823156A (en) * | 2018-07-04 | 2018-11-16 | 陕西神州生物技术有限公司 | For the clinical grade human umbilical cord mesenchymal stem cells composite factor of reparation and the preparation method of freeze-dried powder |
| WO2022116001A1 (en) * | 2020-12-01 | 2022-06-09 | 深圳先进技术研究院 | Method for regulating differentiation and paracrine function of mesenchymal stem cells |
| CN114642683A (en) * | 2022-02-25 | 2022-06-21 | 宁波一棵芽生物科技有限公司 | Preparation method of dental pulp mesenchymal stem cell lysate with photoaging resistance |
| US11786556B2 (en) | 2016-11-18 | 2023-10-17 | Power Of Platelets Pte. Ltd. | Method for preparing a growth factors containing platelet releasate |
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2014
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106566802A (en) * | 2016-08-31 | 2017-04-19 | 山东景源生物科技有限公司 | Preparation method for preparing culture kit of umbilical cord blood mesenchemal stem cells |
| CN106434530A (en) * | 2016-11-16 | 2017-02-22 | 沈阳细胞治疗工程技术研发中心有限公司 | Endothelial cell culture solution |
| CN106434530B (en) * | 2016-11-16 | 2019-06-18 | 沈阳细胞治疗工程技术研发中心有限公司 | A kind of culture fluid of endothelial cell |
| US11786556B2 (en) | 2016-11-18 | 2023-10-17 | Power Of Platelets Pte. Ltd. | Method for preparing a growth factors containing platelet releasate |
| CN108823156A (en) * | 2018-07-04 | 2018-11-16 | 陕西神州生物技术有限公司 | For the clinical grade human umbilical cord mesenchymal stem cells composite factor of reparation and the preparation method of freeze-dried powder |
| WO2022116001A1 (en) * | 2020-12-01 | 2022-06-09 | 深圳先进技术研究院 | Method for regulating differentiation and paracrine function of mesenchymal stem cells |
| CN114642683A (en) * | 2022-02-25 | 2022-06-21 | 宁波一棵芽生物科技有限公司 | Preparation method of dental pulp mesenchymal stem cell lysate with photoaging resistance |
| CN114642683B (en) * | 2022-02-25 | 2023-10-10 | 宁波一棵芽生物科技有限公司 | Preparation method of dental pulp mesenchymal stem cell lysate with photoaging resistance |
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