CN102367435A - Preparation of human platelet-rich plasma and application of same in isolation and culture of human mesenchymal stem cells - Google Patents
Preparation of human platelet-rich plasma and application of same in isolation and culture of human mesenchymal stem cells Download PDFInfo
- Publication number
- CN102367435A CN102367435A CN2011103444640A CN201110344464A CN102367435A CN 102367435 A CN102367435 A CN 102367435A CN 2011103444640 A CN2011103444640 A CN 2011103444640A CN 201110344464 A CN201110344464 A CN 201110344464A CN 102367435 A CN102367435 A CN 102367435A
- Authority
- CN
- China
- Prior art keywords
- under
- plasma
- preparation
- blood
- people
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000004623 platelet-rich plasma Anatomy 0.000 title claims abstract description 44
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 9
- 238000002955 isolation Methods 0.000 title abstract 3
- 210000002381 plasma Anatomy 0.000 claims abstract description 23
- 210000004369 blood Anatomy 0.000 claims abstract description 14
- 239000008280 blood Substances 0.000 claims abstract description 14
- 238000000926 separation method Methods 0.000 claims abstract description 14
- 239000000203 mixture Substances 0.000 claims abstract description 12
- 238000005119 centrifugation Methods 0.000 claims abstract description 7
- 210000004700 fetal blood Anatomy 0.000 claims description 10
- 238000009987 spinning Methods 0.000 claims description 8
- 230000000740 bleeding effect Effects 0.000 claims description 7
- 210000001113 umbilicus Anatomy 0.000 claims description 7
- 238000010128 melt processing Methods 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 229920002123 Pentastarch Polymers 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 229940101738 pentastarch Drugs 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 238000000034 method Methods 0.000 abstract description 15
- 230000000694 effects Effects 0.000 abstract description 9
- 239000003102 growth factor Substances 0.000 abstract description 7
- 239000002994 raw material Substances 0.000 abstract description 5
- 239000013049 sediment Substances 0.000 abstract 2
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 210000003743 erythrocyte Anatomy 0.000 abstract 1
- 235000012041 food component Nutrition 0.000 abstract 1
- 238000010257 thawing Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 16
- 210000001772 blood platelet Anatomy 0.000 description 11
- 230000004069 differentiation Effects 0.000 description 8
- 210000005259 peripheral blood Anatomy 0.000 description 8
- 239000011886 peripheral blood Substances 0.000 description 8
- 210000003995 blood forming stem cell Anatomy 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 6
- 210000003954 umbilical cord Anatomy 0.000 description 5
- 238000012258 culturing Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000002093 peripheral effect Effects 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 230000017423 tissue regeneration Effects 0.000 description 4
- 210000000988 bone and bone Anatomy 0.000 description 3
- 210000004748 cultured cell Anatomy 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000011134 hematopoietic stem cell transplantation Methods 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 230000000050 nutritive effect Effects 0.000 description 3
- 230000004072 osteoblast differentiation Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- 102100022464 5'-nucleotidase Human genes 0.000 description 2
- 208000034423 Delivery Diseases 0.000 description 2
- 102100037241 Endoglin Human genes 0.000 description 2
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 2
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 2
- 208000009329 Graft vs Host Disease Diseases 0.000 description 2
- 102000006354 HLA-DR Antigens Human genes 0.000 description 2
- 108010058597 HLA-DR Antigens Proteins 0.000 description 2
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 2
- 101000678236 Homo sapiens 5'-nucleotidase Proteins 0.000 description 2
- 101000881679 Homo sapiens Endoglin Proteins 0.000 description 2
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 2
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 description 2
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 2
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 description 2
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 2
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 210000000845 cartilage Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 2
- 229960003957 dexamethasone Drugs 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 208000024908 graft versus host disease Diseases 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005374 membrane filtration Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 206010048282 zoonosis Diseases 0.000 description 2
- APIXJSLKIYYUKG-UHFFFAOYSA-N 3 Isobutyl 1 methylxanthine Chemical compound O=C1N(C)C(=O)N(CC(C)C)C2=C1N=CN2 APIXJSLKIYYUKG-UHFFFAOYSA-N 0.000 description 1
- JKYKXTRKURYNGW-UHFFFAOYSA-N 3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-sulfonic acid Chemical compound O=C1C2=CC=CC=C2C(=O)C2=C1C(O)=C(O)C(S(O)(=O)=O)=C2 JKYKXTRKURYNGW-UHFFFAOYSA-N 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 1
- 108010081589 Becaplermin Proteins 0.000 description 1
- 208000019838 Blood disease Diseases 0.000 description 1
- -1 CD79a Proteins 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 235000007926 Craterellus fallax Nutrition 0.000 description 1
- 240000007175 Datura inoxia Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 101100400378 Mus musculus Marveld2 gene Proteins 0.000 description 1
- 208000028389 Nerve injury Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 208000009527 Refractory anemia Diseases 0.000 description 1
- 206010072684 Refractory cytopenia with unilineage dysplasia Diseases 0.000 description 1
- 206010039710 Scleroderma Diseases 0.000 description 1
- 206010039966 Senile dementia Diseases 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- 101150099105 alien gene Proteins 0.000 description 1
- 210000002821 alveolar epithelial cell Anatomy 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000001174 ascending effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000037182 bone density Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 208000014951 hematologic disease Diseases 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 208000018706 hematopoietic system disease Diseases 0.000 description 1
- 230000002607 hemopoietic effect Effects 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 150000002475 indoles Chemical class 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 206010025135 lupus erythematosus Diseases 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 230000008764 nerve damage Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 238000009832 plasma treatment Methods 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 201000003068 rheumatic fever Diseases 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 230000037314 wound repair Effects 0.000 description 1
Images
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides preparation of human platelet-rich plasma and application of the same in isolation and culture of human mesenchymal stem cells. According to the invention, erythrocytes and a mixture of leucocytes and platelets are separated from human whole blood through centrifugation, then centrifugation with a centrifugal force greater than a centrifugal force used in above-mentioned centrifugation is carried out on residual blood plasma, obtained sediment is collected and is subjected to freezing-thawing treatment respectively at a temperature no more than -20 DEG C and a temperature no less than normal temperature, and the sediment is removed through separation so as to obtain human platelet-rich plasma. The invention has the following beneficial effects: raw materials used in the method are widely available; the blood resource is fully utilized; the obtained product of platelets is purer, contains more abundant nutritional components including growth factors and has a more ideal effect when used for isolation and culture of human mesenchymal stem cells.
Description
Technical field
The present invention relates in the biomedical sector the preparation of people's platelet rich plasma and the application in the human mesenchymal stem cell separation and Culture; Especially can ordinary method include but not limited to people's platelet rich plasma of supplying the human mesenchymal stem cell separation and Culture to use by having separated discarded blood plasma behind red corpuscle and white corpuscle and the hematoblastic mixture, particularly still be underutilized at present and depleted human cord blood/blood plasma is raw material, further having prepared.
Background technology
Mescenchymal stem cell (MSCs) is a kind of multipotent adult stem cells with self and multinomial differentiation potential.MSCs separates in the middle of marrow obtain, and external adherent growth is fusiformis.Except can be to mesoblastic skeletonization, become fat and to become the cartilage direction to divide outside the pale of civilization, MSCs is proved to be and can also strides germinal layer and be divided into the various kinds of cell pedigree, like liver cell, pneumonocyte, muscle cell, neurocyte etc.MSCs does not express HLA II class antigen, only expresses the HLA I class antigen of minute quantity, and its immunogenicity is extremely low, and is therefore low to joining the type requirement in Transplanted cells.MSCs distributes in human body extensively, can from the multiple tissue of human body, separate at present to obtain, and has comprised tissues such as peripheral blood, fatty tissue, Cord blood, umbilical cord, placenta.
MSCs can provide suitable microenvironment for hemopoietic stem cell (HSCs), and multinomial research shows, the existence of MSCs has promoted the reconstruction of hemopoietic system after propagation and the HSCT of HSCs.Compare with single HSCT, MSCs and hemopoietic stem cell co-transplantation can significantly improve treatment of diseases effects such as white blood disease and refractory anemia.In addition, MSCs also has immunoloregulation function, can suppress the propagation and the activation of immunocytes such as bone-marrow-derived lymphocyte, T lymphocyte, DC cell.Therefore; MSCs can prevent and treat the graft versus host disease (GVHD) that produces after the allosome HSCT; Avoid the immunological rejection that in organ transplantation, produces, and treat spontaneous immunological disease, like lupus erythematosus, rheumatic arthritis and scleroderma etc.Because MSCs has characteristics such as propagation, differentiation and reduced immunogenicity; Thereby can be used as organizational project ideal seed cell source; Carry out tissue repair and regeneration; And be used for the damage of multiple disease or the reparation of diseased organ, like cardiovascular and cerebrovascular diseases, hepatopathy, bone and muscle decline property disease, brain and marrow nerve injury, senile dementia etc., obtained significant curative effect.MSCs is easy to import and expression alien gene, also is a kind of good carrier of gene therapy.Therefore, MSCs has huge application potential and very wide application prospect.
MSCs need be through external cultivation and amplification, to reach the cell quantity of clinical application.Need to add the foetal calf serum (FBS) of a certain amount of (10 ~ 20%) in the traditional M SCs culture system, as the source of nutritive ingredients such as growth factor, trace element.But use the cellular product of animal serum production often to exist problems: FBS batch differences bigger, reduced the repeated and stable of cell cultures; Exist recessive allele to pollute and immunoreactive risk, after cultured cells transplanting, found the proteic antibody of anti-FBS (Heiskanen A et al., Stem Cells. 2007 with FBS like Heiskanen etc. and Sundin etc.; 25; 197-202; Sundin M et al., Haematologica. 2007; 92:1208-15).In addition, FBS also can cause the change of the Physiology and biochemistry and the metabolic characteristic of culturing cell, as causing in the MSCs culturing process to the differentiation of cartilage direction (Yokoyama M et al., J Biosci Bioeng. 2008; 106; 46-50) etc.Particularly, Chang Keneng contains the infectious agent and unknown zoonosis that is made up of protein in the animal serum, can cause propagating the more higher threat of zoonosis.
People's platelet rich plasma (PRP) is meant and with respect to whole blood, contains the hematoblastic blood plasma of higher level.PRP is existing in plastic sugery at present to be used widely, is mainly used in the bone that promotes after the bone grafting and merges and improve bone density, wound repair and be used for soft tissue repair etc.Contain the multiple growth factor that can keep MSCs propagation among the PRP, like Thr6 PDGF BB (PDGFs), β-transforming growth factor (TGF-β), Urogastron (EGF), Basic Fibroblast Growth Factor (bFGF) etc.And along with PC improves, the corresponding raising of the concentration of this type growth factor.Become the PRP of peripheral body successfully to be used for adipose-derived and vitro culture (Kocaoemer A et al., Stem Cells. 2007 derived from bone marrow MSCs; 25:1270-1278; Schallmoser K et al., Transfusion 2007; 47:1436-1446).
The preparation of PRP at present, like methods such as Landesberg method and Aghaloo methods, principle all is to utilize the difference of each density of fraction size in the blood, to people's whole blood first with once or the centrifugation of gradation isolate red corpuscle and white corpuscle and hematoblastic mixture.For example, gradation mode can adopt ascending cf-isolate respectively red corpuscle (as under cf-≤200g centrifugal 10 minutes) and white corpuscle and hematoblastic mixture (as under cf-200 ~ 900g centrifugal 10 minutes).After the processing, thrombocyte and white corpuscle mainly all are enriched in the platelet rich plasma layer, because of platelet content lower (platelet poor plasma), go out of use usually in the remaining blood plasma.At present the platelet rich plasma that obtains of these methods because thrombocyte is separately difficult with white corpuscle, is difficult to realize thrombocyte and leukocytic utilization respectively, and the high density white corpuscle of sneaking in the rich platelet then might play spinoff for tissue repair etc.On the one hand, be difficult to a large amount of acquisitions, and the collection of peripheral blood also can bring misery to donor, on the other hand, become peripheral body also contaminated because of contact easily like exogenous factors such as radiation, viruses because the blood source of peripheral blood is nervous.
Summary of the invention
To above-mentioned situation; The present invention provides a kind of preparation method of people's platelet rich plasma; The method that peripheral blood that goes out of use after particularly separating according to conventional hemopoietic stem cell and/or umbilical cord blood plasma further prepare PRP, and with these application of resultant PRP in including but not limited to the human mesenchymal stem cell separation and Culture.
The preparation method of people's platelet rich plasma of the present invention; Be after people's whole blood is isolated red corpuscle and white corpuscle and hematoblastic mixture with centrifugation; Residue blood plasma again with greater than before the mode spinning and the collecting precipitation thing of maximum centrifugal force; With the throw out of collecting respectively at≤0 ℃ with the condition that is not less than normal temperature under freeze-melt processing after, separate disgorging, obtain people's platelet rich plasma.
When carrying out red corpuscle and white corpuscle and hematoblastic mixture separation; Preferably in people's whole blood, add earlier as isocyatic hydroxyethylamyle of 6% (w/v) or the isocyatic pentastarch of 10% (w/v) commonly used; To the system the whole content of its weight/volume be 1 ~ 2% and full and uniform mixing after; Separating red corpuscle under≤200g cf-respectively successively; With under 200 ~ 900g cf-, separate white corpuscle and hematoblastic mixture, under＞900g cf-, residue blood plasma is separated then.
Further, to the more preferably mode of said residue plasma treatment, be under >=1000g cf-, to carry out spinning in the aforesaid method.
In the aforesaid method; Said the throw out of being collected by residue blood plasma spinning is frozen-melts when handling; Preferred mode be respectively-20 ℃ and 35 ℃ ~ 42 ℃, be preferably under 37 ℃ the condition and freeze-melt processing at least twice repeatedly; Then greater than to said should the residue plasma separation cf-under, throw out is wherein removed in spinning.To separating the supernatant of disgorging, according to circumstances or need, further filtration treatment, for example, can be through≤0.22 μ m membrane filtration.Resulting people's platelet rich plasma is to be advisable by usual manner cryopreservation (for example≤-20 ℃).
Among the above-mentioned preparation method of the present invention, said people's whole blood, but except that the end user became peripheral body, particularly preferably being with the human cord blood was raw material, prepared corresponding people's bleeding of the umbilicus platelet rich plasma (cbPRP).Though its preparation process and condition etc. are as good as, the source of people's bleeding of the umbilicus horn of plenty more not only can make the bleeding of the umbilicus that is not fully utilized as yet at present and popularizes along with what umbilical cord blood hematopoietic stem cell stored; By a large amount of remaining Cord blood blood plasma behind the separating umbilical blood hemopoietic stem cell; Can be fully utilized, become the good source of bleeding of the umbilicus PRP, and test-results shows; White corpuscle concentration contained in the platelet rich plasma that obtains thus is lower than 1/20 than ordinary method, and thrombocyte is purer.Especially with respect to becoming peripheral body, the source of Cord blood is more original, contains the abundant more nutritive ingredient that comprises growth factor.
Test-results proves, in-vitro separation, cultivation that people's platelet rich plasma (PRP) that method for preparing of the present invention is obtained and/or people's bleeding of the umbilicus platelet rich plasma (cbPRP) are used for human mesenchymal stem cell can have better effect.
Be appreciated that; The above-mentioned people's platelet rich plasma of the present invention preparing method's raw material sources are abundant; Blood resource is utilized more fully, reduced the waste of resource, resulting thrombocyte is also purer; And contain the abundanter nutritive ingredient that comprises growth factor, for the relevant cell product that avoids the use of the production of FBS substratum can cause the risk of clinical application effective solution route is provided.
Below in conjunction with the embodiment of accompanying drawing and embodiment, foregoing of the present invention is remake further detailed description.But should this be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following instance.Do not breaking away under the above-mentioned technological thought situation of the present invention, various replacements or change according to ordinary skill knowledge and customary means are made all should comprise within the scope of the invention.
Description of drawings
Fig. 1 is the 5% PRP (PC 1000 * 10 with the inventive method preparation
9Individual/L) the microscopically state that climbs out of around the tissue block of the MSCs of culture medium culturing.
Fig. 2 the present invention is with the MSCs surface marker characteristic of the PRP cultivation of preparation.
Fig. 3 is the microscopically state that the MSCs of employing PRP cultivation of the present invention has normal Osteoblast Differentiation ability.
Fig. 4 is the microscopically state that the MSCs of employing PRP cultivation of the present invention has normal one-tenth fat differentiation capability.
Embodiment
Embodiment 1
Press usual manner, people's adult blood sample of gathering is originally added 6% hydroxyethylamyle to its content in system be ~ 1% (w/v) and abundant mixing.Centrifugal 6 minutes of 70g removes lower floor's red corpuscle.Remainder centrifugal 11 minutes with 420g separates obtaining " thrombocyte+white corpuscle " mixture layer, can be used for the storage of hemopoietic stem cell.Remaining platelet poor plasma layer centrifugal 10 minutes in 1000g to the pipe end, is inhaled platelet aggregation to remove most of supernatant; To improve hematoblastic concentration; After resuspended, use Backman Coulter Ac.T Diff2 cellanalyzer to measure PC, then it is taken out-20 ℃ of freezing backs respectively; Put immediately in 37 ℃ of water-baths, continue to rock and make it mixing, melt fast.Freeze so repeatedly-melt twice after, remove cell debris with centrifugal 15 minutes of 3000g, obtain PRP.As required, this PRP also can be again with 0.22 μ m membrane filtration, in-20 ℃ of preservations.
Embodiment 2
Cord blood with full-term normal delivery or caesarean healthy puerpera's umbilical cord is a raw material, presses the same manner operation of embodiment 1, obtains corresponding cbPRP.
Embodiment 3
The cbPRP that obtains with embodiment 2 carries out the culture experiment of MSCs.
The cultivation of umbilical cord MSCs:
Umbilical cord is taken from full-term normal delivery or caesarean healthy puerpera, is put in after the collection to contain 4 ℃ of preservations in two anti-PBS solution, handles in the 48h.Umbilical cord is removed blood vessel, shreds to 1mm
3About.The tissue block of every 1.5cm umbilical cord is inoculated in 1 90mm Tissue Culture Dish, with containing DMEM-LG/F12 (1
:1, v/v) add 10 (the v) %FBS and (the v) % cbPRP (PC 1000 * 10 that adds 5
9Individual/L) substratum, place 37 ℃, the CO of volume(tric)fraction 5%
2Cultivate in the saturated humidity incubator.According to the cell growing state, every 3-5 days full dose is changed liquid once.The result is as shown in Figure 1, and cell climbs out of around tissue block.When treating that cell reaches 70% degree of converging, with pancreatin/EDTA digestion of 0.05 (w/v) %, then by 10000/cm
2By the density inoculation of going down to posterity.In the culturing process that goes down to posterity, full dose was changed liquid in per 3 days, when attached cell reaches 90% degree of converging, repeated aforesaid operations and went down to posterity.
Different PCs are to the influence of MSCs propagation:
The MSCs cell of FBS substratum separation and Culture is with 10000/cm
2Density be inoculated in the 6 porocyte culture plates; After inoculation under same cbPRP concentration and the different blood platelet concentration conditions, after the 1st, 2,3,4 day, respectively MSCs is gathered in the crops counting, with 10%FBS contrast, the influence that relatively different PCs are bred MSCs among the cbPRP.Wherein, adopt 3% cbPRP of the present invention, PC difference 1200,800,400,100 (* 10
9Individual/L) and the substratum of 3% centrifuged supernatant, MSCs is in the Yield (unit: ten thousand) of different time points.The result is as shown in table 1.Experiment shows that PC is 800 ~ 1200 * 10
9Individual/during L, the MSCs expanding effect is best.
Table 1 PC is to the influence of MSCs propagation
| PC | |
Day 1 | Day 2 | Day 3 | Day 4 |
| Supernatant | 10.0 | 9.8 | 23.6 | 28.6 | 33.9 |
| 100 | 10.0 | 9.0 | 30.2 | 47.3 | 56.5 |
| 400 | 10.0 | 10.4 | 35.4 | 65.3 | 98.2 |
| 800 | 10.0 | 9.2 | 40.4 | 80.7 | 102.6 |
| 1200 | 10.0 | 9.9 | 39.1 | 79.2 | 107.9 |
| FBS | 10.0 | 8.5 | 22.7 | 54.2 | 62.4 |
Different cbPRP concentration are to the influence of MSCs propagation:
The MSCs cell of FBS substratum separation and Culture is with 10000/cm
2Density be inoculated in the 6 porocyte culture plates, confirming under the condition of PC the comparison of different cbPRP concentration and peripheral blood PRP, 10%FBS effect aspect MSCs propagation.After after the inoculation the 1st, 2,3,4 day, MSCs is gathered in the crops counting equally.
Adopt the substratum of the different cbPRP concentration of the present invention, and 3 (v) when % peripheral blood PRP, 10%FBS, the Yield of each time point MSCs (unit: ten thousand).The result is as shown in table 2.Experiment shows, when cbPRP concentration>3% the time, culture effect is best, and obviously be better than the culture effect of 10%FBS substratum.Same concentration (3%) cbPRP is better than the culture effect of peripheral blood PRP substratum.
The different PRP concentration of table 2 are to the influence of MSCs propagation
| | Day | 0 | Day 1 | Day 2 | Day 3 | Day 4 |
| 0% cbPRP | 10.0 | 7.8 | 12.7 | 11.0 | 9.1 | |
| 1% cbPRP | 10.0 | 9.1 | 29.3 | 50.2 | 70.4 | |
| 2% cbPRP | 10.0 | 9.2 | 28.3 | 69.5 | 78.3 | |
| 3% cbPRP | 10.0 | 6.7 | 32.4 | 67.1 | 119.2 | |
| 4% cbPRP | 10.0 | 8.5 | 33.4 | 67.2 | 120.5 | |
| 5% cbPRP | 10.0 | 8.9 | 38.3 | 74.7 | 126.3 | |
| 7.5% cbPRP | 10.0 | 8.3 | 30.1 | 63.6 | 127.2 | |
| 10% cbPRP | 10.0 | 10.2 | 33.5 | 64.9 | 124.7 | |
| 3% peripheral blood PRP | 10.0 | 7.0 | 30.7 | 42.8 | 101.1 | |
| 10%FBS | 10.0 | 7.4 | 22.6 | 54.0 | 63.0 |
The MSCs surface marker detects:
After the P5 of cbPRP separation and Culture digested for cell routine, with CD105, CD73, the CD79a of PE mark, the CD90 of FITC mark, CD14, CD34, CD45, HLA-DR antibody and corresponding homotype contrasting marking cell thereof carried out streaming and detect.The result is as shown in Figure 2: the cbPRP cultured cells is expressed CD73, CD90, and CD105 does not express CD14, CD34, CD45, CD79a, HLA-DR meets the characteristic of MSCs surface marker.
Analytical Chemical Experiment:
FBS or cbPRP cultured cells are respectively with 10000/cm
2Density be inoculated in 24 orifice plates, when waiting to grow to 70% degree of converging, in substratum, add differentiation inductor.The Osteoblast Differentiation inductor comprises 100 nM DEXAMETHASONE BP98,2 mM b-phospho-glycerols, and and 50 μ M xitix were changed a subculture in per 3 days, during 3 all left and right sides, dyeed with sodium alizarinsulfonate.Become the fat differentiation inductor to comprise 0.5 mM 1-methyl-3-isobutyl--xanthine, the hot beautiful and 0.5 μ M DEXAMETHASONE BP98 of 50 μ M indoles was changed a subculture, and about 2 weeks, was dyeed with oil red in per 3 days.Result such as Fig. 3 and shown in Figure 4 show that the MSCs that cbPRP cultivates has normal skeletonization and becomes the fat differentiation capability.Wherein, the pearl among Fig. 3 is the MSCs Osteoblast Differentiation part of dyeing back demonstration.The MSCs that pearl shows for the dyeing back among Fig. 4 becomes fat differentiation part.
Claims (9)
1. the preparation method of people's platelet rich plasma; After it is characterized in that people's whole blood isolated red corpuscle and white corpuscle and hematoblastic mixture with centrifugation; Residue blood plasma again with greater than before the mode spinning and the collecting precipitation thing of maximum centrifugal force; With the throw out of collecting respectively at≤0 ℃ with the condition that is not less than normal temperature under freeze-melt processing after, separate disgorging, obtain people's platelet rich plasma.
2. preparation method as claimed in claim 1; It is characterized in that in people's whole blood adding in hydroxyethylamyle or pentastarch to the system the whole content of its weight/volume and be 1 ~ 2% and full and uniform mixing after; Separating red corpuscle under≤200g cf-respectively successively; Under 200 ~ 900g cf-, separate white corpuscle and hematoblastic mixture, under＞900g cf-, separate then remaining blood plasma.
3. preparation method as claimed in claim 2 is characterized in that said to carrying out under residue being separated in of blood plasma >=1000g cf-.
4. like the described preparation method of one of claim 1 to 3; It is characterized in that said to the throw out collected by residue blood plasma spinning freezing repeatedly under the condition of-20 ℃ and 35 ~ 42 ℃ respectively-melt processing at least twice after; Under greater than cf-to said residue plasma separation, centrifugal disgorging.
5. the preparation method of people's bleeding of the umbilicus platelet rich plasma; After it is characterized in that human cord blood isolated red corpuscle and white corpuscle and hematoblastic mixture with centrifugation; Residue blood plasma again with greater than before the mode spinning and the collecting precipitation thing of maximum centrifugal force; With the throw out of collecting respectively at≤0 ℃ with the condition that is not less than normal temperature under freeze-melt processing after, separate disgorging, obtain people's bleeding of the umbilicus platelet rich plasma.
6. preparation method as claimed in claim 5; It is characterized in that in human cord blood adding in hydroxyethylamyle or pentastarch to the system the whole content of its weight/volume and be 1 ~ 2% and full and uniform mixing after; Separating red corpuscle under≤200g cf-successively; Under 200 ~ 900g cf-, separate white corpuscle and hematoblastic mixture, under＞900g cf-, separate then remaining blood plasma.
7. preparation method as claimed in claim 6 is characterized in that said to carrying out under residue being separated in of blood plasma >=1000g cf-.
8. like the described preparation method of one of claim 5 to 7; It is characterized in that said to the throw out collected by residue blood plasma spinning freezing repeatedly under the condition of-20 ℃ and 35 ~ 42 ℃ respectively-melt processing at least twice after; Under greater than cf-to said residue plasma separation, centrifugal disgorging.
9. the application of the described people's platelet rich plasma of one of claim 1 to 8 in the human mesenchymal stem cell separation and Culture.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110344464 CN102367435B (en) | 2011-11-04 | 2011-11-04 | Preparation of human platelet-rich plasma and application of same in isolation and culture of human mesenchymal stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110344464 CN102367435B (en) | 2011-11-04 | 2011-11-04 | Preparation of human platelet-rich plasma and application of same in isolation and culture of human mesenchymal stem cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102367435A true CN102367435A (en) | 2012-03-07 |
| CN102367435B CN102367435B (en) | 2013-01-16 |
Family
ID=45760028
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110344464 Active CN102367435B (en) | 2011-11-04 | 2011-11-04 | Preparation of human platelet-rich plasma and application of same in isolation and culture of human mesenchymal stem cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102367435B (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103655614A (en) * | 2012-09-19 | 2014-03-26 | 臻景生物技术（上海）有限公司 | Application of adipose tissue progenitor cell for preventing or treating hepatitis |
| CN103861088A (en) * | 2014-03-04 | 2014-06-18 | 奥思达干细胞有限公司 | Stem cell preparation for treating primary liver cancer and preparation method thereof |
| CN107937334A (en) * | 2017-11-01 | 2018-04-20 | 杭州易文赛生物技术有限公司 | A kind of cell factor extract and its application |
| CN109078224A (en) * | 2018-09-14 | 2018-12-25 | 四川新生命干细胞科技股份有限公司 | Skin injury repairing material and preparation method thereof |
| CN109082409A (en) * | 2018-09-14 | 2018-12-25 | 四川新生命干细胞科技股份有限公司 | A kind of fat stem cell that tissue repairing ability is strong is separately cultured and screening technique |
| CN109288868A (en) * | 2018-10-31 | 2019-02-01 | 广州市天河诺亚生物工程有限公司 | M-PRP preservation method, preparation method and application |
| CN109303759A (en) * | 2018-11-23 | 2019-02-05 | 北京安溢生物科技有限公司 | A kind of preparation method and applications of Cord blood platelet rich plasma |
| CN110538196A (en) * | 2019-08-13 | 2019-12-06 | 杭州易文赛生物技术有限公司 | Platelet-rich plasma and method for extracting platelet-rich plasma |
| CN111303270A (en) * | 2020-02-18 | 2020-06-19 | 浙江中医药大学 | A kind of method for preparing platelet growth factor concentrate by using set |
| CN113046317A (en) * | 2021-03-31 | 2021-06-29 | 河南省遗传资源细胞库有限公司 | Method for extracting platelet-rich plasma based on preparation of umbilical cord blood hematopoietic stem cells |
| CN113924361A (en) * | 2019-05-22 | 2022-01-11 | 善克思腾(股份)责任有限公司 | Method for expanding adult stem cells from whole blood |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI643953B (en) * | 2014-12-11 | 2018-12-11 | 訊聯生物科技股份有限公司 | Method for culturing progenitor cells by autologous plasma |
-
2011
- 2011-11-04 CN CN 201110344464 patent/CN102367435B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| 孙洁等: "富血小板血浆制备方法的研究进展", 《国际口腔医学杂志》 * |
| 张洪涛等: "富血小板血浆对人骨髓间充质干细胞成骨诱导的影响", 《中国组织工程研究与临床康复》 * |
| 王悦等: "富血小板血浆提取方法的探讨", 《实用口腔医学杂志》 * |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103655614B (en) * | 2012-09-19 | 2019-06-28 | 西比曼生物科技（上海）有限公司 | The application in hepatitis is being prevented or treated to adipose tissue progenitor cell |
| CN103655614A (en) * | 2012-09-19 | 2014-03-26 | 臻景生物技术（上海）有限公司 | Application of adipose tissue progenitor cell for preventing or treating hepatitis |
| CN103861088A (en) * | 2014-03-04 | 2014-06-18 | 奥思达干细胞有限公司 | Stem cell preparation for treating primary liver cancer and preparation method thereof |
| CN103861088B (en) * | 2014-03-04 | 2016-03-23 | 奥思达干细胞有限公司 | A kind of stem cell medicine for the treatment of primary hepatocarcinoma and preparation method thereof |
| CN107937334A (en) * | 2017-11-01 | 2018-04-20 | 杭州易文赛生物技术有限公司 | A kind of cell factor extract and its application |
| CN109078224A (en) * | 2018-09-14 | 2018-12-25 | 四川新生命干细胞科技股份有限公司 | Skin injury repairing material and preparation method thereof |
| CN109082409A (en) * | 2018-09-14 | 2018-12-25 | 四川新生命干细胞科技股份有限公司 | A kind of fat stem cell that tissue repairing ability is strong is separately cultured and screening technique |
| CN109288868A (en) * | 2018-10-31 | 2019-02-01 | 广州市天河诺亚生物工程有限公司 | M-PRP preservation method, preparation method and application |
| CN109303759A (en) * | 2018-11-23 | 2019-02-05 | 北京安溢生物科技有限公司 | A kind of preparation method and applications of Cord blood platelet rich plasma |
| CN113924361A (en) * | 2019-05-22 | 2022-01-11 | 善克思腾(股份)责任有限公司 | Method for expanding adult stem cells from whole blood |
| CN110538196A (en) * | 2019-08-13 | 2019-12-06 | 杭州易文赛生物技术有限公司 | Platelet-rich plasma and method for extracting platelet-rich plasma |
| CN111303270A (en) * | 2020-02-18 | 2020-06-19 | 浙江中医药大学 | A kind of method for preparing platelet growth factor concentrate by using set |
| CN113046317A (en) * | 2021-03-31 | 2021-06-29 | 河南省遗传资源细胞库有限公司 | Method for extracting platelet-rich plasma based on preparation of umbilical cord blood hematopoietic stem cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102367435B (en) | 2013-01-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102367435B (en) | Preparation of human platelet-rich plasma and application of same in isolation and culture of human mesenchymal stem cells | |
| Domenis et al. | Adipose tissue derived stem cells: in vitro and in vivo analysis of a standard and three commercially available cell-assisted lipotransfer techniques | |
| Bieback et al. | Clinical protocols for the isolation and expansion of mesenchymal stromal cells | |
| Mohammadi et al. | Differentiation of adipocytes and osteocytes from human adipose and placental mesenchymal stem cells | |
| CN101748096A (en) | Sub totipotential stem cell and preparation method and application thereof | |
| CN108456657B (en) | Canine umbilical cord mesenchymal stem cells and preparation method and cryopreservation method thereof | |
| CN110938590B (en) | Mesenchymal stem cell serum-free medium and application thereof | |
| CN104762257B (en) | A kind of method preparing mescenchymal stem cell from umbilical cord | |
| WO2012068710A1 (en) | Methods for extracting mesenchymal stem cell from slight amount human adipose tissue and mass cultivation thereof | |
| CN105420179A (en) | Method for simultaneously extracting epithelial cells and mesenchymal stem cells from umbilical cord and placenta amnion tissues | |
| CN103695369B (en) | Umbilical cord mesenchymal stem cells vitro culture and amplification method | |
| CN108300690A (en) | A kind of isolated culture method and serum free medium of fat mesenchymal stem cell | |
| CN104762258B (en) | A kind of cultural method of human umbilical cord mesenchymal stem cells | |
| CN1778905B (en) | Separating culture and use for fatty mesenchymal dry cell | |
| Hassan et al. | Isolation of umbilical cord mesenchymal stem cells using human blood derivatives accompanied with explant method | |
| Romanov et al. | Optimized protocol for isolation of multipotent mesenchymal stromal cells from human umbilical cord | |
| Tasma et al. | Production of extracellular vesicles from equine embryo-derived mesenchymal stromal cells | |
| Tevlin et al. | A novel method of human adipose-derived stem cell isolation with resultant increased cell yield | |
| CN107083359B (en) | Stem cell culture medium and stem cell separation method | |
| CN102146359A (en) | Method for extracting original mesenchymal stem cells from placenta and serum-free amplification | |
| CN107287156A (en) | A kind of isolated culture method of fat mesenchymal stem cell and its application | |
| KR101380561B1 (en) | Equine Amniotic Fluid-Derived Multipotent Stem Cells and Method for Producing the Same | |
| CN108728408B (en) | Canine fetal membrane mesenchymal stem cells and preparation method and medium used | |
| JP2007525979A (en) | Mesenchymal progenitor cell serum-free suspension culture system | |
| CN102703380B (en) | Sub-totipotent stem cell, preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |