CN102757935A - Method for extracting hematopoietic stem cell from placental chorionic tissue and construction method of hematopoietic stem cell - Google Patents
Method for extracting hematopoietic stem cell from placental chorionic tissue and construction method of hematopoietic stem cell Download PDFInfo
- Publication number
- CN102757935A CN102757935A CN201110112037XA CN201110112037A CN102757935A CN 102757935 A CN102757935 A CN 102757935A CN 201110112037X A CN201110112037X A CN 201110112037XA CN 201110112037 A CN201110112037 A CN 201110112037A CN 102757935 A CN102757935 A CN 102757935A
- Authority
- CN
- China
- Prior art keywords
- stem cell
- placenta
- cell
- positive
- hemopoietic stem
- 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.)
- Pending
Links
Images
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for extracting a CD133 positive stem cell from a placental chorionic tissue. The method comprises the following steps: (1) collecting a placenta to a placenta collection box, and adding protection fluid into the collection box; (2) carrying out information registration and serological virus detection before the placenta separates the stem cell; (3) washing blood clot and hematocele in the placenta tissue; (4) separating the placenta stem cell by a mechanical method; (5) concentrating and purifying the placenta stem cell, and extracting the CD133 positive stem cell from the placenta; and (6)storing the CD133 positive stem cell. The stored CD133 positive stem cell can be used for the transplanting of a hematopoietic stem cell, angiogenesis, repairing of nerve injury and the like. The invention has the advantages that the CD133 positive stem cell extracting flow is convenient; theCD133 positive stem cell extracting cost is lower; and a whole placenta can be treated rapidly at a time, the placenta contains a large number of mononuclear cells, and the content of CD133 positive stem cells in the placenta reaches 2.0 to 5.0 percent. The technology is also applicable to constructing a CD133 positive stem cell bank, and the CD133 positive stem cells stored in the cell bank can be reused by adults for many times and stored for a long time, and are convenient to use at any time.
Description
Technical field
The present invention relates to a kind of method of from the placental villi membrane tissue, extracting hemopoietic stem cell; Be meant a kind of method of from the placental villi membrane tissue, extracting the positive stem cell of CD133 especially, and utilize the method for the positive stem cell constructing hemopoietic stem cell bank of CD133 that extracts.
Background technology
(Hemopoietic Stem cell, doing HSC) is translated from English " stem " to hemopoietic stem cell, means " tree ", " doing " and " origin ".Be similar to one tree and do and can grow crotch, leaf, and bloom with the result etc.Generally; Hemopoietic stem cell is meant the cell of undeveloped mature still; It is the origin of all hematopoietic cells and immunocyte; It not only can be divided into red corpuscle, white corpuscle and thrombocyte, also can interdepartmental system be divided into the cell of various histoorgans, has self, multidirectional differentiation and goes back to the nest (being directional migration to hemopoietic tissue organ) potential.General hemopoietic stem cell derives from three channels: marrow hemopoietic stem cells; Procedure for peripheral blood stem cells; Umbilical cord blood hematopoietic stem cell.Placenta is the place of fetus and the exchange of mother's blood, contains profuse blood microcirculation.The people is in the interogestate stage of mother, and placenta is one of organ that at first forms.This is illustrated in the growth, atomization of embryonic stem cell, and in placenta, being detained has a large amount of early stage stem cells, and probably these stem cells also continue keeping the ability of hemopoietic stem cell in placenta.
Placenta contains abundant early stage hemopoietic stem cell of each stage, CD34 since finding always as the stem cell surface mark.Its no species specificity, the selective expression also has expression in hemopoietic stem cell at vascular endothelial cell.CD133 is AC133, also is called: Prominin1; Hematopoitic stem cell antigen; Prominin-like 1, is a kind of transmembrane protein, belongs to the Prominin family member; Relative molecular mass is 120ka, [Miraglia S, Godffey W such as Miraglia in 1997; Yin AH; Et al.A novel five trans membrance h ematopoietic stem cell antigen:isolation, characterization, and molecular cl oning [J] .Blood; 1997,90 (12): 5013-5021] it is proposed as new hemopoietic stem cell surface antigen.Different with CD34 antigen is; CD133 is progenitor cell late; As pre B cell, red be colony forming unit (colony forming unit-erythrocyte, CFU-E), grain is that (colony forming unit-granulocyte does not express on CFU-G) colony forming unit.CD133 mainly is expressed in adult, fetus marrow, bleeding of the umbilicus, and on the CD34+ cell subsets of hemopoietic tissues such as tire liver, representative is than CD34+ primary hematopoietic stem subgroup more.2005, the Mikkola of California, USA university professor reported first placenta was a main hemocytopoietic organ (Dev Cell 8:365-75,2005 of fetus; Cell Stem Cell 2:252-263,2008).2009, the mature placenta of people finders such as professor Kuypers was a hemocytopoietic organ (Exp Biol Med, 234:813-823,2009), and this conclusion is confirmed by many laboratories subsequently.The mature placenta of people can provide a large amount of CD133 positive cell and other primary hematopoiesis ancester cells, is fit to human the transplanting.And generally acknowledging that at present CD133 male hemopoietic stem cell is more early stage than the positive hemopoietic stem cell of CD34, it can be 10 times that same source Cord blood can get hemopoietic stem cell that hemopoietic stem cell of being obtained by placenta or the group that turns out form unitary total quantity.The CD133 positive cell has long-term culture-initiating cell (Long-term culture-initiating cells; LTC-IC) and long-term reconstitute hematopoiesis cell (Long term repopulation cells; LTRC) ability; Be primary hematopoietic cell, transplant and to plant work for a long time after giving the host.In addition, discover that recently LTC-IC is mainly seen in the positive subgroup of the positive CD34 of CD133, a small set of CD133 positive/CD34 negative cells also has long-term proliferation potential, so think that CD133 is primitive hematopoietic cell crowd (comprising a CD34 negative cells) sign.Cord blood CD 34 that Rappold etc. did and CD133 sorting discover that the CD34+ cell amplification potential with a sample is lower than the CD133+ cell.Therefore the CD133 sorting possibly be superior to the CD34 sorting.
CD133 can also do as other non-hematopoiesis, the surface marker of progenitor cell, as NSC (neural stem cell, NSC).The experiment in vitro that Stamm etc. carry out confirms; The CD133+ cell in peripheral blood source can be divided into neurocyte, has potential applicability in clinical practice, can be used for the cell therapy of some nervous system disorderss; Comprise Spinal injury, Alzheimer syndrome and parkinson's disease.
The item key that vascular endothelial precursor cell (EPCs) is different from mature endothelial cell is CD133.Find that after deliberation endotheliocyte can not combine the antibody of CD133.The endotheliocyte that confirms differentiation and maturation does not have CD133.These explanations CD133 can be used as the surface marker that EPCs is different from mature endothelial cell.Many scholars sub-elect the CD133 positive cell from Cord blood and marrow, vitro culture confirms to be divided into endotheliocyte.Therefore have the people to propose, since the CD133+ cell can be divided into endotheliocyte external, in vivo tests may improve patient's heart function through revascularization in the myocardial infarction patient so.Got into the clinical study stage at present.
Bonanno etc. discover that clinical grade sorting Cord blood CD133 positive cell can obtain an amount of original hemopoietic forebody cell, and these cells have the potential of very high hematopoiesis activity and external mesenchymal cell.
The method of separating placenta stem-cell at present has mechanical phonograph recorder separation, and lavage, collagenase digestion, mechanical phonograph recorder separation pair cell damage big and separate wastes time and energy, and is unfavorable for industrialization, and lavage is mainly used in angioplast, and cell yield is low; The domestic Zhou Shengli of collagenase digestion etc.
Patent from placenta tissue, extract the novel method (patent No. 01131190.8) that hemopoietic stem cell is used for setting up hemopoietic stem cell bank and mention; It mainly is the tissue-derived CD34 positive cell of sorting; It uses single collagenase digesting placenta tissue 15 minutes; But aforesaid method can only obtain the minute quantity cell, the cell count that cell count obtains well below mechanically sepg.
Summary of the invention
In view of this, main purpose of the present invention is to provide a kind of method of from placenta tissue, extracting the positive stem cell of CD133 simple to operation.From placenta tissue, extract the positive stem cell of CD133 and set up stem cell bank to be used for clinical treatment be main idea of the present invention, realize that this technical scheme is to adopt the multiple digestion method.The multiple digestion technology that the present invention uses can be digested to whole placenta tissue unicellularly fast, and the cell suspension viscosity that obtains is not high, helps concentrating cells.The positive hemopoietic stem cell flow process of the CD133 of extraction of the present invention is convenient, cost is lower, once can fast processing one whole placenta, and amount of mononuclear cells is many, can reach 5-10 * 10
9Individual cell, wherein the positive content of CD133 is up to 2.0-5.0%.This technology also is fit to make up hemopoietic stem cell bank, and the hemopoietic stem cell of being preserved can be used for the adult repeatedly to be used, but long-term storage is convenient to take at any time.
Technical scheme of the present invention is summarized as follows:
(1) gathers placenta and female blood, Cord blood, the sample of gathering is carried out the related diseases pathogenic microorganism detect; With the Information Monitoring input system;
(2) the chorion tissue block is washed with buffer salt solution under aseptic condition repeatedly, removes wherein blood clot and hematocele;
(3) method with enzymic digestion obtains postdigestive mixed solution, and mixed Digestive system is obtained cell suspension with strainer filtering;
(4), and it is changed in the centrifugal bag with adding precipitation agent behind the centrifuging concentrating cells suspension;
(5) with low cf-centrifugal separation the red corpuscle in the mixed solution is separated with tissue block impurity earlier, remove red corpuscle and tissue block impurity;
(6) with the high centrifugal force centrifugal separation nucleated cell in the centrifugal bag is concentrated again, obtain to be rich in the mixture of CD133 positive cell;
(7) the placenta hemopoietic stem cell in the centrifugal bag is transferred in frozen pipe/bag, and adds frozen protective material;
(8) will be added with frozen protectant placenta hemopoietic stem cell cooling, it is temporary to put into the liquid nitrogen temporary library;
(9) cell after separating is carried out HLA and join type, CD133 positive cell content detection, monocyte count, intracellular toxin and microorganism detection detection, change qualified cell over to the liquid nitrogen prolonged preservation, and with the relevant information input system.
According to aforesaid method, we have reached following effect (1) and can handle whole placenta; (2) the treatment time weak point had only about 3 hours, farthest kept cell characteristics; (3) cell quantity is many, can reach 5-10 * 10
9Individual/placenta; (4) the CD133 positive rate is high, reaches 3.0-6.0%, can satisfy the adult fully and repeatedly use; (5) biggest advantage of the present invention is the direct positive stem cell of the CD133 of enrichment sufficient amount from placenta tissue.
Description of drawings
Fig. 1 is the chorion derived cell immunophenotype analytical results figure after separating;
Fig. 2 is streaming phenotype analytical figure after the CD133 sorting of the membrane derived CD133 positive cell of placental villi;
Fig. 3 is that the membrane derived CD133 positive cell of placental villi is cultivated back cell CD31 phenotype detection figure with the endothelium division culture medium;
Fig. 4 is that the positive ratio of the people CD45 of the membrane derived CD133 positive cell of placental villi reconstitute hematopoiesis in NOD/SCID mouse body detects figure.
Embodiment
Embodiment one: the separation of placenta derived cell
1. the collection of placenta is gathered placenta in the situation of puerpera's informed consent.Wash placenta with SPSS, then placenta is packed in the special-purpose aseptic collection box, and add and gather liquid.Subsequently 4 ℃ on placenta is transported to the experiment place, the time of transportation placenta is no more than 48 hours.
2. the separation of placental villi membrane tissue hemopoietic stem cell: the chorion tissue is isolated with the method for machinery in (1); (2) tissue is shredded the cubic centimetre to 0.1-1, then with aqueous phosphate solution repeatedly the washed tissue piece make tissue block not have tangible blood clot and other foreign material more than 5 times; (3) according to adding the mixture of collagenase, pancreatin and Unidasa and DNA enzyme with the ratio of tissue volume than 1: 1,37 ℃ of constant temperature digestions 60 minutes; (4) hydroxyethylamyle and Digestive system 1: 6 add, with three bag centrifugation method, and centrifugal 10 minutes earlier with 50g, abandon red corpuscle, and then with 500g centrifugal 10 minutes, obtain the chorion histocyte, wherein be rich in CD133 positive stem cell; (5) the chorion histocyte is resuspended to 45 volumes with protection liquid, adds 5 milliliters DMSO therein, then with 4 parts of 0.5 milliliter/pipe samplings, portion is used for counting, and several parts keep sample and do correlation detections such as joining type in addition; To remain 48 ml cells suspensions and change frozen bag over to, preserve through changing in the liquid nitrogen behind the programmed cooling.
3. flow cytometry analysis.Cell after separating is pressed each sample 1 * 10
6Cell is in charge of, and after the method for knowing by the professional person is used antibody labeling, is resuspended to the 400 μ L streaming pipe of packing into PBS.Detect, analyze with the FACS Calibur of BD company stream type cell analyzer.
Result as shown in Figure 1: our isolated cells is mainly derived from placenta tissue (having only 2.36% hemocyte); Isolated cells crowd comprises the CD133 positive cell of total cell 4.04%, the positive and CD105 male mescenchymal stem cell of the CD73 of total cell about 20%.
Embodiment two:
The sorting of the membrane derived CD133 positive cell of placental villi:
Sorting method only is changed to the placental villi membrane tissue with the source with embodiment one.With the cell after the sorting,, manage out the CD133 positive cell from placenta derived cell branch through the magnetic bead sorting method of using the professional person all to know.
Result shown in Figure 2: behind the CD133 magnetic bead sorting, the CD133 positive cell purity of acquisition is 93.07% of total cell.
Embodiment three:
The membrane derived CD133 positive cell of placental villi is divided into endotheliocyte
Result shown in Figure 3: with the positive ratio of the cell after the mescenchymal stem cell culture medium culturing (middle figure) CD31 is 2.85%, is 74.36% with the positive ratio of the cell after the endotheliocyte culture medium culturing (right figure) CD31.
Embodiment four:
The membrane derived cell of placental villi is rebuild the hemopoietic system of irradiation back NOD/SCID mouse
With the membrane derived cell of the vigorous resuspended placental villi of arteries and veins power A solution, adjustment cell concn 5x10
7Individual/milliliter (high dose group) and 1x10
7Individual/milliliter (low dose group).Press table 1 animal is divided into groups, import the cell or the vigorous arteries and veins power A solution of the corresponding dosage of 100 microlitres through the tail vein.After cell implanted for 6 weeks, get survival mice marrow and separate mononuclearcell, detect expressing human CD45 in marrow and the peripheral blood with flow cytometer
+Cell count detects the membrane derived CD133 positive cell implantation of the placental villi situation of transplanting.
Result shown in Figure 4: people's CD45 positive cell ratio in NOD/SCID mouse marrow, the high dose group implantation rate: 83.73 ± 23.20%, low dose group implantation rate: 29.83 ± 13.01%; Middle figure is that the highest implantation rate of high dose group is the highest implantation rate of low dose group with right figure.
The above is merely preferred embodiment of the present invention, and is in order to restriction the present invention, not all within spirit of the present invention and principle, any modification of being done, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. method of from the placental villi membrane tissue, extracting hemopoietic stem cell may further comprise the steps:
Gather placenta;
Method with machinery is processed into tissue block with the placental villi film;
Under aseptic condition, said tissue block is washed repeatedly with aqueous phosphate solution;
Obtain postdigestive mixed solution with the said tissue block of enzymic digestion, mixed solution is filtered obtain cell suspension;
Shift out blood plasma with centrifuging, and the concentrating cells suspension, the mixture of hemopoietic stem cell is rich in acquisition.
2. method according to claim 1 is characterized in that, said hemopoietic stem cell is the positive stem cell of CD133.
3. method according to claim 1 is characterized in that, the placenta of gathering has been carried out the pathogenic micro-organism detection.
4. method according to claim 1; It is characterized in that said centrifuging is two step centrifuging, promptly removes red corpuscle and tissue block impurity in the mixed solution with low cf-earlier; With high centrifugal force the hemopoietic stem cell in the centrifugal bag is concentrated again, obtain to be rich in the mixture of CD133 positive cell;
5. method according to claim 1 is characterized in that, also comprises storage method, is about to the said mixture that is rich in hemopoietic stem cell and is transferred in the frozen pipe, adds frozen protective material; To be added with frozen protectant hemopoietic stem cell cooling, and put into liquid nitrogen and store.
6. hemopoietic stem cell that obtains according to any one method of claim 1-5.
7. a method of utilizing the described hemopoietic stem cell of claim 6 to build hemopoietic stem cell bank is characterized in that, also is included in to separate preceding Information Monitoring input system with placenta;
Detect separating the back hemopoietic stem cell, change qualified hemopoietic stem cell over to the liquid nitrogen prolonged preservation, and with the method for relevant information input system.
8. method according to claim 7 is characterized in that, said hemopoietic stem cell is the positive stem cell of CD133.
9. method according to claim 7 is characterized in that, said detection comprises that HLA joins type, CD133 positive cell content detection, monocyte count, intracellular toxin and microorganism detection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110112037XA CN102757935A (en) | 2011-04-29 | 2011-04-29 | Method for extracting hematopoietic stem cell from placental chorionic tissue and construction method of hematopoietic stem cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110112037XA CN102757935A (en) | 2011-04-29 | 2011-04-29 | Method for extracting hematopoietic stem cell from placental chorionic tissue and construction method of hematopoietic stem cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102757935A true CN102757935A (en) | 2012-10-31 |
Family
ID=47052587
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110112037XA Pending CN102757935A (en) | 2011-04-29 | 2011-04-29 | Method for extracting hematopoietic stem cell from placental chorionic tissue and construction method of hematopoietic stem cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102757935A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104212761A (en) * | 2014-08-20 | 2014-12-17 | 北京瑞思德生物科技有限公司 | Kit for preparing placenta stem cells |
| CN104739865A (en) * | 2015-02-13 | 2015-07-01 | 杭州易文赛生物技术有限公司 | Method for preparing placenta hematopoietic stem cell preparation |
| CN109652372A (en) * | 2019-01-09 | 2019-04-19 | 陕西九州细胞基因工程有限公司 | A kind of quick separating of human placenta source candidate stem cell, preparation method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1407088A (en) * | 2001-09-06 | 2003-04-02 | 周胜利 | Method for establishing hematopoietic stem cells bank by extracting hematopoietic cells from placenta tissues |
| CN101483998A (en) * | 2006-05-11 | 2009-07-15 | 武部直子 | Methods for collecting and using placenta cord blood stem cells |
-
2011
- 2011-04-29 CN CN201110112037XA patent/CN102757935A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1407088A (en) * | 2001-09-06 | 2003-04-02 | 周胜利 | Method for establishing hematopoietic stem cells bank by extracting hematopoietic cells from placenta tissues |
| CN101483998A (en) * | 2006-05-11 | 2009-07-15 | 武部直子 | Methods for collecting and using placenta cord blood stem cells |
Non-Patent Citations (2)
| Title |
|---|
| 刘佳云 等: "人胎盘CD133+细胞具有高增殖潜能集落形成细胞特性", 《解剖学报》, vol. 39, no. 6, 31 December 2008 (2008-12-31) * |
| 章涛 等: "人胎盘组织造血干/祖细胞的分离富集", 《中国实验血液学杂志》, vol. 14, no. 5, 30 October 2006 (2006-10-30) * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104212761A (en) * | 2014-08-20 | 2014-12-17 | 北京瑞思德生物科技有限公司 | Kit for preparing placenta stem cells |
| CN104739865A (en) * | 2015-02-13 | 2015-07-01 | 杭州易文赛生物技术有限公司 | Method for preparing placenta hematopoietic stem cell preparation |
| CN104739865B (en) * | 2015-02-13 | 2018-12-18 | 杭州易文赛生物技术有限公司 | A method of preparing placental hematopoietic stem cell preparation |
| CN109652372A (en) * | 2019-01-09 | 2019-04-19 | 陕西九州细胞基因工程有限公司 | A kind of quick separating of human placenta source candidate stem cell, preparation method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhu et al. | Manual isolation of adipose-derived stem cells from human lipoaspirates | |
| CN104164403B (en) | Method for extracting and culturing adipose-derived stem cells | |
| CN101421393B (en) | Methods and compositions for enhancing engraftment of hematopoietic stem cells | |
| CN107236704B (en) | From the method for placenta separating mesenchymal stem cell and the digestive enzyme compositions used | |
| CN101638637B (en) | Kit for processing human marrow, cord blood and peripheral blood cells and cell processing method | |
| CN108633877A (en) | A kind of human umbilical cord mesenchymal stem cells excretion body freeze-dried powder and its method of preparation | |
| CN102676451B (en) | Method for separating mesenchymal stem cells from placenta | |
| CN105238752A (en) | Culture system and culture method for efficient amplification in vitro of autologous NK cells | |
| CN102851254A (en) | Procurement, isolation, and cryopreservation of endometrial/menstrual cells | |
| Amirkhani et al. | A rapid sonication based method for preparation of stromal vascular fraction and mesenchymal stem cells from fat tissue | |
| CN103667187A (en) | Isolated culture method of human adipose-derived stem cells and construction method of stem cell bank | |
| CN110628708A (en) | Separation and purification method of high-purity pig muscle stem cells | |
| CN104711220A (en) | Novel method for preparing menstrual blood mesenchymal stem cells | |
| CN105238748A (en) | Preparation method of placenta-source decidua parietalis mesenchymal stem cells by separation and refrigeration | |
| CN107299082A (en) | Placenta interstitial cell and the method for being trained mescenchymal stem cell are separated from tissue | |
| CN103966162A (en) | Novel menstrual blood-derived mesenchymal stem cell separation method | |
| CN102604892A (en) | Stem cell sample density separating medium and stem cell separation method for human marrow, umbilical cord blood or peripheral blood | |
| CN102154200B (en) | Preparation and storage of mesenchymal stem cells for clinical treatment | |
| CN107653225A (en) | A kind of culture medium and its amplification method for being used to expand human mesenchymal stem cell | |
| CN104877965B (en) | A kind of method for preparing mature erythrocyte | |
| CN104523753A (en) | Preparation method, product and application of human umbilical cord mesenchymal stem cell cultural supernatant active factor and cell lysis buffer | |
| CN104762258B (en) | A kind of cultural method of human umbilical cord mesenchymal stem cells | |
| CN106801032A (en) | The construction method of people's amnioic epithelium stem cell bank | |
| CN103695369A (en) | Umbilical cord mesenchymal stem cell in-vitro culture and amplification method | |
| CN104152409A (en) | Method for simultaneous isolated culture of canine bone marrow mesenchymal stem cells and multifunctional hematopoietic stem cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20121031 |