CN103352026A - Method for cultivating autologous umbilical cord mesenchymal stem cells by adopting human umbilical cord blood rich platelet lysate - Google Patents
Method for cultivating autologous umbilical cord mesenchymal stem cells by adopting human umbilical cord blood rich platelet lysate Download PDFInfo
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Abstract
The invention relates to a method for cultivating autologous umbilical cord mesenchymal stem cells by adopting human umbilical cord blood rich platelet lysate. The conventional cell bank preparing processes adopt blood serum of calves or fetal calves to cultivate, digest, cryopreserve and the like, so that the risk of heterogeneous protein residue certainly exists in the application of clinic umbilical cord stem cells in the future. The method comprises the following steps of umbilical cord blood rich platelet serum preparation, platelet lysate preparation, preparation of serous without platelet, the confirmation of the content of cell factors, namely PDGF-AB, FGF2, TGF-Beta and VFGF in the platelet lysate, the separation and the primary culture of umbilical cord stem cells, the culture and passage of umbilical cord stem cells, the cryopreservation of umbilical cord stem cells, and the karyotype analysis. The method is used for cultivating stem cells.
Description
Technical field:
The present invention relates to regenerative medicine and biological technical field, be specifically related to the comprehensive utilization of rich platelet serum in the Cord blood and from the non-animal derived production system of body mescenchymal stem cell.
Background technology:
In China, the development of stem cell human bank day by day by vast reproduction age family paid close attention to, when most pregnant woman are chosen as child and store Cord blood, also can select the umbilical cord stem cell is cultivated and preserves.Present cell bank preparation process adopts all that calf or foetal calf serum are cultivated, digestion, the process such as frozen, therefore, really can have the residual risk of foreign protein in the clinical umbilical cord stem cell in future is used.
Mescenchymal stem cell is the stem cell that a class has self, propagation and multi-lineage potential, the external adherent fibroblast-like cells that is, can be divided into bone, cartilage and adipocyte, can secrete have in a large number immunomodulatory, bioactive cytokine and the somatomedin such as hematopoiesis support, promotion vasculogenesis, chemotactic tissue injury reparation.
Mescenchymal stem cell (MSCs) is to derive from the mesoblastic stem cell that multinomial differentiation capability is arranged, although more with content in the marrow, but studies show that, in its hetero-organization of people and organ, existence is arranged, as adult peripheral blood, fat, skin, bleeding of the umbilicus, umbilical cord even placental villi and amniotic fluid in also all exist, particularly have the MSCs of a large amount of high-qualitys in umbilical cord, placenta and the fat.In marrow, umbilical cord and the fatty tissue, contain maximum MSCs in fat and the umbilical cord, and the amplification ability of MSCs is the strongest in the umbilical cord, and the MSCs in three sources can break up skeletonization and cartilage (Kern etc., 2006) as broad as long aspect morphology and the immunogenicity and three.On the paracrine function, umbilical cord MSCs is better than bone marrow MSCs far away, and the multiple somatomedin of umbilical cord MSCs secretion is more than 1,000 times of bone marrow MSCs (Friedman etc., 2007).It approached in etap and embryonic stem cell, the many biological properties that possess again multipotential stem cell, use and need not join type, quantity is abundant, differentiation capability is strong, orientablely be divided into the multiple stem cells such as vascular stem cell, epithelial stem cell, neural stem cell, liver stem cells, do not produce tumour simultaneously, do not have ethnics Problem.
Analyze thus, research adopts the MSCs in fertility source should have more broad prospect of application and more excellent result for the treatment of.It is advantageous that: itself be the fertility waste, the source is sufficient, easily obtains; The cell age is light, differentiation capability is strong, does not have other adult stems to be subject to the infringements such as environment, radiation, and quality is pure; The stem cell paracrine is powerful, and therefore nutrition adjusting and the immunoregulation effect to the body performance is stronger than other adult stem cells.
The present bibliographical informations of process such as the cultivation of MSCs, amplification, storage mainly are to adopt the method for foetal calf serum (Sarugaser etc. 2005; Sotiropoulou etc. 2006).The umbilical cord mesenchymal stem cells and the amplification quantity that adopt foetal calf serum 5 ~-20% easily to obtain are large, produce and also more easily carry out commercialization control (Friedman etc. 2007).MSCs is present in the multiple adult tissue, can separate at present obtaining from the tissues such as umbilical cord, placenta, Adult Human Bone Marrow, peripheral blood, fat.Yet, in these known sources, MSCs content average out to 1/10
8~ 1/10
3The needed MSCs quantity of common clinical cell therapy is 10
6~ 10
9Individual, in order to obtain the MSCs of sufficient amount, need to carry out vitro culture and go down to posterity amplification separating the MSCs that obtains.Commonly used contain animal serum (FBS or FCS) substratum and can in 17 ~ 30 days, increase MSCs to satisfy the needs of application.But adopt animal serum to exist two kinds of risks, the firstth, heterology animal source protein residue pollutes reaction, the 2nd, the pathogen contamination such as the virus that potential animal source is known or unknown, mycoplasma to the allergy that the treatment body produces.
Summary of the invention:
The object of the invention is to overcome the prior art deficiency, solve the needs of following clinical application, provide a kind of without animal serum, without foreign protein from somatic umbilicus mescenchymal stem cell cultural method.
The object of the present invention is achieved like this:
A kind of human cord blood rich platelet lysate is cultivated from somatic umbilicus mescenchymal stem cell method, the steps include: the preparation of Cord blood rich platelet serum, the preparation of thrombocyte lysate, the dehematize preparation of platelet serum, cytokine PDGF-AB in the thrombocyte lysate, FGF2, TGF-β, determining of VEGF content, the separation of umbilical cord stem cell and former culture, the cultivation of umbilical cord stem cell and go down to posterity the umbilical cord stem cell cryopreserving, chromosome karyotype analysis, the signature analysis of umbilical cord mesenchymal stem cells after cultivating.
Described human cord blood rich platelet lysate is cultivated from somatic umbilicus mescenchymal stem cell method, the preparation of described Cord blood rich platelet serum: the Cord blood 50 ~ 120ml of collection is divided in the aseptic centrifuge tube of 50ml, under 4 ~ 30 degree temperature, centrifugal 5 ~ 25 minutes of 150 ~ 220g, the upper strata is for being rich in thrombocyte serum, adopt the heparin of 2000 ~ 5000UI to make antithrombotics in blood taking bag or the heparin tube, the serum on upper strata accounts for 40% of cumulative volume.
Described human cord blood rich platelet lysate is cultivated from somatic umbilicus mescenchymal stem cell method, the preparation of described thrombocyte lysate: rich platelet serum is moved in the new aseptic centrifuge tube, adopt Sysmex hematology analyzer (KX--21) that contained red corpuscle, white corpuscle, platelet counts in whole blood and the serum are counted, umbilical cord mesenchymal stem cells is cultivated with hematoblastic quantity desirable in the serum 0.5 ~ 1 * 10
9Between/the ml; Serum was placed-80 degree refrigerators frozen 2 ~ 24 hours; Contain thrombocyte serum behind 37 degree rapid fluid resuscitations after frozen, through 800 ~ 1200g centrifugal 5 ~ 30 minutes, remove cell debris, supernatant liquor is the thrombocyte lysate, carry out proportioning with its certain concentration after, be used for the cultivation from the somatic umbilicus stem cell.
Described human cord blood rich platelet lysate is cultivated from somatic umbilicus mescenchymal stem cell method, the preparation of the described platelet serum of dehematizing: after the Cord blood centrifugation obtains the process of upper strata rich platelet serum, to staying middle level karyocyte in the centrifuge tube and lower floor's red corpuscle with 800g ~ 1000g speed centrifugal 30 ~ 40 minutes, the limpid serum layer in upper strata is the platelet serum of dehematizing.
Described human cord blood rich platelet lysate is cultivated from somatic umbilicus mescenchymal stem cell method, the cytokine PDGF-AB in the described thrombocyte lysate, FGF2, TGF-β, the determining of VEGF content: adopt R﹠amp; The enzyme linked immunological of d system (ELISA) test kit detects.
Described human cord blood rich platelet lysate is cultivated from somatic umbilicus mescenchymal stem cell method, the separation of described umbilical cord stem cell and former culture: it is long that the term fetus umbilical cord is got 15 ~ 20cm, after physiological saline and 75% alcohol wash, be cut into segment, respectively artery and vein are peeled off, the magnificent Tong Shi glue of centre is separated, under aseptic condition, dissected into 0.2 ~ 2mm small tissue blocks, directly in Tissue Culture Flask, cultivate; Or through 0.1% ~ 0.2% 37 lower collagenase digestings of degree 1 hour, obtain single cell suspension, counting is regulated cell concn, then is inoculated in the sterile culture flask, places 37 ℃, 5%CO
2Cultivate in the incubator; Adopt basic medium DMEM to add the autologous platelet lysate and cultivate, the adding proportion of lysate is decided according to original platelet counts, generally between 5% ~ 20%.
Described human cord blood rich platelet lysate is cultivated from somatic umbilicus mescenchymal stem cell method, the cultivation of described umbilical cord stem cell and going down to posterity: former culture changed liquid once in per 3 ~ 5 days, when umbilical cord mesenchymal stem cells growth degrees of fusion reaches 80%, cell culture fluid is collected, after 200g was centrifugal, supernatant liquor was for subsequent use; Cell in the culturing bottle washes twice through the aseptic PBS without calcium ions and magnesium ions, with 0.25% pancreatin, 37 degree digestion 2 ~ 3 minutes, after cell presents circular and progressively breaks away from the culturing bottle bottom, above-mentioned cell culture supernatant for subsequent use is poured in the culturing bottle of digestion, stop the pancreatin reaction; Blow and beat gently postdigestive umbilical cord stem cell and collection with transfer pipet, centrifugal 5 minutes of 200g increases the cell renewed vaccination of collecting, until reach the cell quantity of expection in new culturing bottle.
Described human cord blood rich platelet lysate is cultivated from somatic umbilicus mescenchymal stem cell method, and described umbilical cord stem cell cryopreserving: the main component of frozen storing liquid comprises the DMSO of basic medium 5 ~ 10% and removes autologous platelet serum; Adopt the trysinization way, umbilical cord mesenchymal stem cells is digested from culturing bottle, with 10
6Individual stem cell/ml concentration is suspended in the above-mentioned frozen storing liquid and with cell suspension and is placed in the 2.0ml cryopreservation tube, and cryopreservation tube cools to-80 degree gradually in Virahol is the program freezing storing box of medium, then places the medium-term and long-term preservation of-150 degree liquid nitrogen gaseous environments.
Described human cord blood rich platelet lysate is cultivated from somatic umbilicus mescenchymal stem cell method, described chromosome karyotype analysis: in the cell mitogen Metaphase Chromosome sample preparation, colchicine processings, hypotonic processing, fixing, film-making, the dyeing of Giemsa dye liquor are read the sheet analysis under the mirror afterwards.
Described human cord blood rich platelet lysate is cultivated from somatic umbilicus mescenchymal stem cell method, described mescenchymal stem cell is that described mescenchymal stem cell separation method comprises collagenase dissociation or tissue block partition method from the mescenchymal stem cell in somatic umbilicus source.Described mescenchymal stem cell contains PC 0.5 ~ 1 * 10 in cultivating in body rich platelet serum
9/ ml, the ratio of thrombocyte lysate in substratum is 5 ~ 20%; Described mescenchymal stem cell goes down to posterity in the process, adopts and is with autoserous substratum to carry out the pancreatin neutralizing effect; Comprise in the described mesenchymal stem cell cryopreserving liquid 5 ~ 10% from body without the thrombocyte serum of umbilical cord blood.
Beneficial effect:
1. rich platelet serum of the present invention can be the by product behind the separation Cord blood monokaryon stem cell, and therefore this method does not affect collection, the storage to cord blood stem cell.
2. the present invention fully utilizes the by product Cord blood rich platelet serum lysate in the cord blood stem cell separation, umbilical cord mesenchymal stem cells after the cultivation meets the surface antigen feature of stem cell check, the characteristics of CD90+, CD105+, CD73+, CD34-, CD45-, CD14-, CD79a-, HLA-DR-, and can form normal colony.
3. the present invention adopts from somatic umbilicus blood, under precise time control, Cord blood separated and obtain to be rich in thrombocyte serum, serum is carried out cracking, and a large amount of VEGF, the FGF2 that discharges in the thrombocyte after the cracking, PDGF--AB, the somatomedins such as TGF--β, EGF are replenished the under-nutrition in the serum and are cultivated and the amplification needs in order to satisfy from the somatic umbilicus stem cell.
4. the present invention adopts pancreatin neutralization and the stem cell cryopreserving of autoserum stem cell in going down to posterity, and guarantees there is not allosome in the whole production process, the protein contamination in xenogenesis source.In the body substratum, without any foreign protein and nutritional factor, guarantee the pure of the stem cell goods that obtain, be not subjected to the threat of any allos pathogenic micro-organism, do not have allergy and get rid of the generation of reaction.Get involved without any allosome and foreign sera in the culturing process, be specially adapted to the preparation that following clinical treatment is used stem cell.
5. contain the abundant growing nutrient factor in the Cord blood thrombocyte of the present invention, it is directly carried out cracking after, somatomedin is directly released in the serum, and does not need complicated platelet activation process, namely economy is quick again.
6. the present invention utilizes the by product rich platelet serum of Cord blood sepn process to take full advantage of, and carries out the cultivation of umbilical cord mesenchymal stem cells, and the product of acquisition can farthest meet the following clinical needs without allosome serum, foreign protein in body is used.
7. the present invention's cell of cultivating acquisition keeps the feature of adult stem cell to have: the cell attachment growth, and the morphology aspect shows spindle shape, and growth presents the whirlpool shape; Surface antigen is expressed and to be presented CD73, CD90, CD105 positive expression, CD14, CD34, CD45, CD79a, the HLA-DR expression that is negative; Cell proterties after the cultivation is stable, does not have chromosomal variation to produce.
Description of drawings:
Accompanying drawing 1 is that the present invention adopts the umbilical cord stem cell figure that obtains as substratum from somatic umbilicus blood rich platelet lysate.
Accompanying drawing 2 is that the umbilical cord mesenchymal stem cells surface antigen CD105 after the present invention cultivates expresses the positive.
Accompanying drawing 3 is that the umbilical cord mesenchymal stem cells surface antigen CD90 after the present invention cultivates expresses the positive.
Accompanying drawing 4 is that the umbilical cord mesenchymal stem cells surface antigen CD73 after the present invention cultivates expresses the positive.
Accompanying drawing 5 is that the umbilical cord mesenchymal stem cells surface antigen CD45 after the present invention cultivates expresses feminine gender.
Accompanying drawing 6 is that the umbilical cord mesenchymal stem cells surface antigen CD34 after the present invention cultivates expresses feminine gender.
Accompanying drawing 7 is that the umbilical cord mesenchymal stem cells surface antigen CD14 after the present invention cultivates expresses feminine gender.
Accompanying drawing 8 is that the umbilical cord mesenchymal stem cells surface antigen CD79a after the present invention cultivates expresses feminine gender.
Accompanying drawing 9 is that the umbilical cord mesenchymal stem cells surface antigen HLA-DR after the present invention cultivates expresses feminine gender.
Accompanying drawing 10 is that the present invention carries out chromosome karyotype analysis to the umbilical cord mesenchymal stem cells that is cultured to P2.
Embodiment:
Embodiment 1:
A kind of human cord blood rich platelet lysate is cultivated from somatic umbilicus mescenchymal stem cell method, the steps include: the preparation of Cord blood rich platelet serum, the preparation of thrombocyte lysate, the dehematize preparation of platelet serum, cytokine PDGF-AB in the thrombocyte lysate, FGF2, TGF-β, determining of VEGF content, the separation of umbilical cord stem cell and former culture, the cultivation of umbilical cord stem cell and go down to posterity the umbilical cord stem cell cryopreserving, chromosome karyotype analysis, the surface antigen analysis of umbilical cord mesenchymal stem cells after cultivating.
Embodiment 2:
Cultivate from somatic umbilicus mescenchymal stem cell method according to embodiment 1 described human cord blood rich platelet lysate, the preparation of described Cord blood rich platelet serum: the Cord blood 50 ~ 120ml of collection is divided in the aseptic centrifuge tube of 50ml, under 4 ~ 30 degree temperature, centrifugal 5 ~ 25 minutes of 150 ~ 220g, the upper strata is for being rich in thrombocyte serum, adopt the heparin of 2000 ~ 5000UI to make antithrombotics in blood taking bag or the heparin tube, the serum on upper strata accounts for 40% of cumulative volume.
Embodiment 3:
Cultivate from somatic umbilicus mescenchymal stem cell method according to embodiment 1 or 2 described human cord blood rich platelet lysates, the preparation of described thrombocyte lysate: rich platelet serum is moved in the new aseptic centrifuge tube, adopt Sysmex hematology analyzer (KX-21) that contained red corpuscle, white corpuscle, platelet counts in whole blood and the serum are counted, umbilical cord mesenchymal stem cells is cultivated with hematoblastic quantity desirable in the serum 0.5 ~ 1 * 10
9Between/the ml; Serum was placed-80 degree refrigerators frozen 2 ~ 24 hours; Contain thrombocyte serum behind 37 degree rapid fluid resuscitations after frozen, through 800 ~ 1200g centrifugal 5 ~ 30 minutes, remove cell debris, supernatant liquor is the thrombocyte lysate, carry out proportioning with its certain concentration after, be used for the cultivation from the somatic umbilicus stem cell.
Embodiment 4:
Cultivate from somatic umbilicus mescenchymal stem cell method according to embodiment 1 or 2 or 3 described human cord blood rich platelet lysates, the preparation of the described platelet serum of dehematizing: after the Cord blood centrifugation obtains the process of upper strata rich platelet serum, to staying middle level karyocyte in the centrifuge tube and lower floor's red corpuscle with 800g ~ 1000g speed centrifugal 30 ~ 40 minutes, the limpid serum layer in upper strata is the platelet serum of dehematizing.
Embodiment 5:
Cultivate from somatic umbilicus mescenchymal stem cell method the cytokine PDGF-AB in the described thrombocyte lysate, FGF2, TGF-β, the determining of VEGF content: adopt R﹠amp according to embodiment 1 or 2 or 3 or 4 described human cord blood rich platelet lysates; The enzyme linked immunological of d system (ELISA) test kit detects, and the nutritional factor analysis in the Cord blood thrombocyte lysate sees the following form 1.
Table 1
| Platelet counts | （530±58）×10 6 /ml |
| FGF2 | 204±35 pg/ml |
| TGF-B | 92257±4879 pg/ml |
| VEGF | 650±86 pg/ml |
| PDGF-AB | 19 1000±29010 pg/ml |
Embodiment 6:
Cultivate from somatic umbilicus mescenchymal stem cell method according to embodiment 1 or 2 or 3 or 4 or 5 described human cord blood rich platelet lysates, the separation of described umbilical cord stem cell and former culture: it is long that the term fetus umbilical cord is got 15 ~ 20cm, after physiological saline and 75% alcohol wash, be cut into segment, respectively artery and vein are peeled off, the magnificent Tong Shi glue of centre is separated, under aseptic condition, dissected into 0.2 ~ 2mm small tissue blocks, directly in Tissue Culture Flask, cultivate; Or through 0.1% ~ 0.2% 37 lower collagenase digestings of degree 1 hour, obtain single cell suspension, counting is regulated cell concn, then is inoculated in the sterile culture flask, places 37 ℃, 5% CO
2Cultivate in the incubator; Adopt basic medium DMEM to add the autologous platelet lysate and cultivate, the adding proportion of lysate is decided according to original platelet counts, generally between 5% ~ 20%.
Embodiment 7:
Cultivate from somatic umbilicus mescenchymal stem cell method according to embodiment 1 or 2 or 3 or 4 or 5 or 6 described human cord blood rich platelet lysates, the cultivation of described umbilical cord stem cell and going down to posterity: former culture changed liquid once in per 3 ~ 5 days, when umbilical cord mesenchymal stem cells growth degrees of fusion reaches 80%, cell culture fluid is collected, after 200g was centrifugal, supernatant liquor was for subsequent use; Cell in the culturing bottle washes twice through the aseptic PBS without calcium ions and magnesium ions, with 0.25% pancreatin, 37 degree digestion 2 ~ 3 minutes, after cell presents circular and progressively breaks away from the culturing bottle bottom, above-mentioned cell culture supernatant for subsequent use is poured in the culturing bottle of digestion, stop the pancreatin reaction; Blow and beat gently postdigestive umbilical cord stem cell and collection with transfer pipet, centrifugal 5 minutes of 200g increases the cell renewed vaccination of collecting, until reach the cell quantity of expection in new culturing bottle.
Embodiment 8:
Cultivate from somatic umbilicus mescenchymal stem cell method according to embodiment 1 or 2 or 3 or 4 or 5 or 6 or 7 described human cord blood rich platelet lysates, described umbilical cord stem cell cryopreserving: the main component of frozen storing liquid comprises the DMSO of basic medium 5 ~ 10% and removes autologous platelet serum; Adopt the trysinization way, umbilical cord mesenchymal stem cells is digested from culturing bottle, with 10
6Individual stem cell/ml concentration is suspended in the above-mentioned frozen storing liquid and with cell suspension and is placed in the 2.0ml cryopreservation tube, and cryopreservation tube cools to-80 degree gradually in Virahol is the program freezing storing box of medium, then places the medium-term and long-term preservation of-150 degree liquid nitrogen gaseous environments.
Embodiment 9:
Cultivate from somatic umbilicus mescenchymal stem cell method according to embodiment 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 described human cord blood rich platelet lysates, described chromosome karyotype analysis: in the cell mitogen Metaphase Chromosome sample preparation, colchicine processings, hypotonic processing, fixing, film-making, the dyeing of Giemsa dye liquor are read the sheet analysis under the mirror afterwards.
Embodiment 10:
Cultivate from somatic umbilicus mescenchymal stem cell method according to embodiment 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 described human cord blood rich platelet lysates, the cell that described cultural method obtains keeps the feature of adult stem cell: the cell attachment growth, the morphology aspect shows spindle shape, and growth presents the whirlpool shape; Surface antigen is expressed and to be presented CD73, CD90, CD105 positive expression, CD14, CD34, CD45, CD79a, the HLA-DR expression that is negative; Cell proterties after the cultivation is stable, does not have chromosomal variation to produce.
Embodiment 11:
Cultivate from somatic umbilicus mescenchymal stem cell method according to embodiment 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 described human cord blood rich platelet lysates, described mescenchymal stem cell is that described mescenchymal stem cell separation method comprises collagenase dissociation or tissue block partition method from the mescenchymal stem cell in somatic umbilicus source.Described mescenchymal stem cell contains PC 0.5 ~ 1 * 10 in cultivating in body rich platelet serum
9/ ml, the ratio of thrombocyte lysate in substratum is 5 ~ 20%; Described mescenchymal stem cell goes down to posterity in the process, adopts and is with autoserous substratum to carry out the pancreatin neutralizing effect; Comprise in the described mesenchymal stem cell cryopreserving liquid 5 ~-10% from body without the thrombocyte serum of umbilical cord blood.
Embodiment 12:
Cultivate from somatic umbilicus mescenchymal stem cell method the preparation of (1) Cord blood rich platelet serum according to the described human cord blood rich platelet of above-described embodiment lysate: prepare blood taking bag, wherein place anticoagulant heparin agent 2000UI; Behind mature c-section fetal birth, ligature of the cord remains in Cord blood 96.7ml in umbilical cord and the placenta from what umbilical vein gathered, softly mixes while gathering, prevents because heparin and blood mix inequality and the blood coagulation phenomenon occurs; Sample is delivered in the separating experiment chamber in 1 hour; Between the aseptic preparation of Cord blood, the blood in the collection bag is divided in two aseptic centrifuge tubes of 50ml; Under 4 degree, 150 ~-centrifugal 20 minutes of 220g, be divided into up and down two-phase in the centrifuge tube; Do not stirring under the erythrocytic prerequisite to lower floor, as much as possible the upper strata is being rich in the thrombocyte serum transfers to new aseptic centrifuge tube, approximately 40ml; Obtain PC, 452 * 10 after adopting the analysis of KX--21 haemanalysis instrument
6/ ml.
(2) dehematize red corpuscle layer that platelet serum preparation: 55ml separates through thrombocyte in desk centrifuge, set under the rotating speed 1000g centrifugal 30 minutes; Supernatant liquor 8ml is transferred in the aseptic 15ml centrifuge tube;-80 degree are preserved stand-by.
(3) preparation of Cord blood thrombocyte lysate: 44ml is contained 521 * 10
6/ ml rich platelet serum places-80 degree refrigerators suddenly to freeze 10 hours; After this is frozen contain thrombocyte serum 37 the degree 5 minutes in rapid fluid resuscitation; On platform in the whizzer, through 900g centrifugal 30 minutes, remove the cell debris of precipitation, obtain 43ml Cord blood thrombocyte lysate; Adopt respectively R﹠amp; The enzyme linked immunological kit of the restructuring personnel antibody sandwich of D company, the content of nutritional factor in the analytical pyrolysis liquid, reading absorbancy calculating PDGF-AB at 570nm is 170ng/m, and FGF2 is 210pg/m, and TGF-β is 105ng/m, and VEGF is 567pg/ml; Frozen stand-by in-80 degree refrigerators after the packing.
(4) tissue block method carries out the cultivation from the somatic umbilicus stem cell: the c-section fetal cord is got 15cm, in delivery room, with stroke-physiological saline solution flushing 2 times, 75% alcohol wash 1 time, in the aseptic collection of packing into the suit, is transported to umbilical cord stem cell preparation room; In hundred grades of laminar flow Biohazard Safety Equipments, umbilical cord again after the flushing of physiological saline and alcohol, is cut into the 3cm segment; Artery and vein in the umbilical cord are removed in aseptic dissection, and the magnificent Tong Shi glue in the umbilical cord is peeled off out; Tissue block is shredded into magnificent Tong Shi glue the small tissue blocks of 0.2mm with clipper for surgical use under aseptic condition; Tissue block is seeded in the Tissue Culture Flask, adds basic medium DMEM and 15% autologous platelet lysate and cultivate; Every 5 days, adopt FAB platelet lysate to change liquid.
(5) enzyme process carries out the cultivation from the somatic umbilicus stem cell: natural labor in October the boy baby divide the puerperium, with ligature of the cord, and after collecting Cord blood, cut umbilical cord 10cm, with stroke-physiological saline solution flushing 2 times, the tire that 75% alcohol swab is carefully removed transfection just, in the aseptic collection of packing into the bottle; 15 degree were transported to umbilical cord stem cell preparation room in 1 hour; The same embodiment of umbilical cord (4) is shredded into 1cm size tissue block, digested 3 hours while shaking through 0.2% type i collagen enzyme, 37 degree; Cell suspension removes and does not digest tissue block after 75 order sterile filtrations after the digestion, obtains single cell suspension; After unicellular thrombocyte lysate with basic medium DMEM/F12+5% after centrifugal suspends, be inoculated in the sterile culture flask, place 37 ℃, 5% CO
2Cultivate in the incubator, liquid was changed once every 3 days in the centre.
(6) going down to posterity and amplification procedure without the umbilical cord mesenchymal stem cells of animal serum: former culture 10 days, process goes down to posterity when umbilical cord mesenchymal stem cells growth degrees of fusion reaches 85%; With being collected in the 15ml centrifuge tube from somatic umbilicus blood thrombocyte cracking substratum 10ml in the culturing bottle, centrifugal 5 minutes of 200g stays supernatant liquor for subsequent use; Cell in the culturing bottle is through without after twice of the aseptic PBS flushing of calcium ions and magnesium ions, adds in the 1ml 0.25% pancreatin 37 degree incubators digestion 2--3 minute in culturing bottle; After cell presents circular and progressively breaks away from the culturing bottle bottom, above-mentioned 10ml for subsequent use is contained autoserous cell culture medium pour in the culturing bottle of digestion, stop the pancreatin reaction; Blow and beat gently postdigestive umbilical cord stem cell and collection with transfer pipet, centrifugal 5 minutes of 200g, with the umbilical cord mesenchymal stem cells collected with the PBS washing, centrifugal after, be suspended in the thrombocyte lysate fresh culture of 10mlDMEM/F12+5%, again according to stem cell 5 * 10
3/ cm
2Concentration be inoculated in the new culturing bottle, increased every 3 days, P2 is for cell such as Fig. 1, the cell attachment growth, the morphology aspect shows spindle shape, and growth presents the whirlpool shape.
(7) autoserum carries out the umbilical cord stem cell cryopreserving: adopt from body and dehematize after platelet serum adds 10% DMSO, be mixed with from body frozen storing liquid 1ml; Grow into the umbilical cord mesenchymal stem cells of P1, adopt that embodiment (6) method digests, after centrifugal, the washing, according to 2 * 10
6/ ml concentration is suspended in above-mentioned being placed in the 2.0ml cryopreservation tube of preparing in the body frozen storing liquid and with cell suspension; Cryopreservation tube cools to-80 degree gradually in Virahol is the program freezing storing box of medium, then place the medium-term and long-term preservation of-150 degree liquid nitrogen gaseous environments.
(8) umbilical cord mesenchymal stem cells chromosome karyotype analysis: umbilical cord mesenchymal stem cells was cultivated P3 generation in 18 days through the autologous platelet lysate; Adopt that the preparation of Metaphase Chromosomes sample adopts that the colchicine of 10ug/ml is processed, twice of methyl alcohol: glacial acetic acid=3:1 is fixing, Giemsa dye liquor dyeing after the film-making; Analyze under the mirror, go out report according to international human cytogenetics naming system; No abnormal, such as accompanying drawing 10.
(9) the surface antigen analysis of umbilical cord mesenchymal stem cells after the autologous platelet lysate is cultivated: adopt flow cytometer, carry out the surface antigen analysis after P2 is digested for umbilical cord mesenchymal stem cells; Such as Fig. 2 ~ 4, express successively and present CD105, CD90, CD73 positive expression, such as Fig. 5 ~ 9, CD45, CD34, CD14, CD79a, the HLA-DR expression that is negative; Meet the umbilical cord mesenchymal stem cells standard that traditional training method obtains.
Claims (10)
1. a human cord blood rich platelet lysate is cultivated from somatic umbilicus mescenchymal stem cell method, it is characterized in that: the steps include: the preparation of Cord blood rich platelet serum, the preparation of thrombocyte lysate, the dehematize preparation of platelet serum, cytokine PDGF-AB in the thrombocyte lysate, FGF2, TGF-β, determining of VEGF content, the separation of umbilical cord stem cell and former culture, the cultivation of umbilical cord stem cell and go down to posterity the umbilical cord stem cell cryopreserving, chromosome karyotype analysis, the signature analysis of umbilical cord mesenchymal stem cells after cultivating.
2. human cord blood rich platelet lysate according to claim 1 is cultivated from somatic umbilicus mescenchymal stem cell method, it is characterized in that: the preparation of described Cord blood rich platelet serum: the Cord blood 50 ~ 120ml of collection is divided in the aseptic centrifuge tube of 50ml, under 4 ~ 30 degree temperature, centrifugal 5 ~ 25 minutes of 150 ~ 220g, the upper strata is for being rich in thrombocyte serum, adopt the heparin of 2000 ~ 5000UI to make antithrombotics in blood taking bag or the heparin tube, the serum on upper strata accounts for 40% of cumulative volume.
3. human cord blood rich platelet lysate according to claim 1 and 2 is cultivated from somatic umbilicus mescenchymal stem cell method, it is characterized in that: the preparation of described thrombocyte lysate: rich platelet serum is moved in the new aseptic centrifuge tube, adopt Sysmex hematology analyzer (KX-21) that contained red corpuscle, white corpuscle, platelet counts in whole blood and the serum are counted, umbilical cord mesenchymal stem cells is cultivated with hematoblastic quantity desirable in the serum 0.5 ~ 1 * 10
9Between/the ml; Serum was placed-80 degree refrigerators frozen 2 ~ 24 hours; Contain thrombocyte serum behind 37 degree rapid fluid resuscitations after frozen, through 800 ~ 1200g centrifugal 5 ~ 30 minutes, remove cell debris, supernatant liquor is the thrombocyte lysate, is used for the cultivation from the somatic umbilicus stem cell.
According to claim 1 and 2 or 3 described human cord blood rich platelet lysates cultivate from somatic umbilicus mescenchymal stem cell method, it is characterized in that: the preparation of the described platelet serum of dehematizing: after the Cord blood centrifugation obtains the process of upper strata rich platelet serum, to staying middle level karyocyte in the centrifuge tube and lower floor's red corpuscle with 800g ~ 1000g speed centrifugal 30 ~ 40 minutes, the limpid serum layer in upper strata is the platelet serum of dehematizing.
According to claim 1 and 2 or 3 or 4 described human cord blood rich platelet lysates cultivate from somatic umbilicus mescenchymal stem cell method, it is characterized in that: the cytokine PDGF-AB in the described thrombocyte lysate, FGF2, TGF-β, the determining of VEGF content: adopt R﹠amp; The enzyme linked immunological of d system (ELISA) test kit detects.
According to claim 1 and 2 or 3 or 4 or 5 described human cord blood rich platelet lysates cultivate from somatic umbilicus mescenchymal stem cell method, it is characterized in that: the separation of described umbilical cord stem cell and former culture: it is long that the term fetus umbilical cord is got 15 ~ 20cm, after physiological saline and 75% alcohol wash, be cut into segment, respectively artery and vein are peeled off, the magnificent Tong Shi glue of centre is separated, under aseptic condition, dissected into 0.2 ~ 2mm small tissue blocks, directly in Tissue Culture Flask, cultivate; Or through 0.1% ~ 0.2% 37 lower collagenase digestings of degree 1 hour, obtain single cell suspension, counting is regulated cell concn, then is inoculated in the sterile culture flask, places 37 ℃, 5% CO
2Cultivate in the incubator; Adopt basic medium DMEM to add the autologous platelet lysate and cultivate, the adding proportion of lysate is decided according to original platelet counts, generally between 5% ~ 20%.
According to claim 1 and 2 or 3 or 4 or 5 or 6 described human cord blood rich platelet lysates cultivate from somatic umbilicus mescenchymal stem cell method, it is characterized in that: the cultivation of described umbilical cord stem cell and going down to posterity: former culture changed liquid once in per 3 ~ 5 days, when umbilical cord mesenchymal stem cells growth degrees of fusion reaches 80%, cell culture fluid is collected, after 200g was centrifugal, supernatant liquor was for subsequent use; Cell in the culturing bottle washes twice through the aseptic PBS without calcium ions and magnesium ions, with 0.25% pancreatin, 37 degree digestion 2 ~ 3 minutes, after cell presents circular and progressively breaks away from the culturing bottle bottom, above-mentioned cell culture supernatant for subsequent use is poured in the culturing bottle of digestion, stop the pancreatin reaction; Blow and beat gently postdigestive umbilical cord stem cell and collection with transfer pipet, centrifugal 5 minutes of 200g increases the cell renewed vaccination of collecting, until reach the cell quantity of expection in new culturing bottle.
According to claim 1 and 2 or 3 or 4 or 5 or 6 or 7 described human cord blood rich platelet lysates cultivate from somatic umbilicus mescenchymal stem cell method, it is characterized in that: described umbilical cord stem cell cryopreserving: the main component of frozen storing liquid comprises the DMSO of basic medium 5 ~ 10% and removes autologous platelet serum; Adopt the trysinization way, umbilical cord mesenchymal stem cells is digested from culturing bottle, with 10
6Individual stem cell/ml concentration is suspended in the above-mentioned frozen storing liquid and with cell suspension and is placed in the 2.0ml cryopreservation tube, and cryopreservation tube cools to-80 degree gradually in Virahol is the program freezing storing box of medium, then places the medium-term and long-term preservation of-150 degree liquid nitrogen gaseous environments.
According to claim 1 and 2 or 3 or 4 or 5 or 6 or 7 or 8 described human cord blood rich platelet lysates cultivate from somatic umbilicus mescenchymal stem cell method, it is characterized in that: described chromosome karyotype analysis: in the cell mitogen Metaphase Chromosome sample preparation, colchicine processings, hypotonic processing, fixing, film-making, the dyeing of Giemsa dye liquor are read the sheet analysis under the mirror afterwards.
According to claim 1 and 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 described human cord blood rich platelet lysates cultivate from somatic umbilicus mescenchymal stem cell method, it is characterized in that: described mescenchymal stem cell is the mescenchymal stem cell from the somatic umbilicus source, and described mescenchymal stem cell separation method comprises collagenase dissociation or tissue block partition method; Described mescenchymal stem cell contains PC 0.5 ~ 1 * 10 in cultivating in body rich platelet serum
9/ ml, the ratio of thrombocyte lysate in substratum is 5 ~ 20%; Described mescenchymal stem cell goes down to posterity in the process, adopts and is with autoserous substratum to carry out the pancreatin neutralizing effect; Comprise in the described mesenchymal stem cell cryopreserving liquid 5 ~ 10% from body without the thrombocyte serum of umbilical cord blood.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101919379A (en) * | 2010-08-06 | 2010-12-22 | 青岛奥克生物开发有限公司 | Frozen stock solution used for freezing and storing umbilical mesenchymal stem cells for long time |
-
2013
- 2013-07-24 CN CN201310313363.6A patent/CN103352026B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101919379A (en) * | 2010-08-06 | 2010-12-22 | 青岛奥克生物开发有限公司 | Frozen stock solution used for freezing and storing umbilical mesenchymal stem cells for long time |
Non-Patent Citations (1)
| Title |
|---|
| 许茹 等: "人血小板裂解液替代胎牛血清促进骨髓间质干细胞的增殖", 《南方医科大学学报》 * |
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