CN105969722A - Extraction and preparation method of placenta pluripotent stem cells - Google Patents
Extraction and preparation method of placenta pluripotent stem cells Download PDFInfo
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- CN105969722A CN105969722A CN201610570529.6A CN201610570529A CN105969722A CN 105969722 A CN105969722 A CN 105969722A CN 201610570529 A CN201610570529 A CN 201610570529A CN 105969722 A CN105969722 A CN 105969722A
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- placenta
- pluripotent stem
- placenta hominis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention discloses an extraction and preparation method of placenta pluripotent stem cells. The extraction and preparation method comprises the following steps: under a sterile condition, obtaining placenta tissues; repeatedly washing with PBS, disinfecting with betadine and sufficiently washing a placenta with a saline solution for at least three times; cutting the placenta tissues into fragments with the size of 1*1*1mm<3> and putting the fragments into a sterile centrifugal tube; digesting the placenta tissue fragments with 0.25% trypsase and 0.2% IV type collagenase; centrifuging and collecting to obtain cell sediment; re-suspending the cell sediment into a fetal calf blood serum alpha-MEM culture medium with the volume percent of 10% and freezing by a conventional method; and carrying out flow cytometry analysis on CD73, CD90 and CD105 cell expression. The placenta is obtained under the sterile condition and the placenta is subjected to a series of pre-treatment including PBS repeated washing, placenta surface disinfection and washing of the placenta with the saline solution, so as to guarantee that the obtained placenta is fresh and conveniently guarantee the activity of the pluripotent stem cells in the placenta tissues.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of Placenta Hominis pluripotent stem cell and extract preparation method.
Background technology
Stem cell can self renewal, and various cell line can be divided into.These cells can be divided into embryonic stem cell (ESC) and non-embryonic stem cells or adult stem cell (ASC).The latter can not be divided into various tissue (multipotency) again, and its growth rate is less than embryonic stem cell.Therefore, the application of embryonic stem cell is better than the application of adult stem cell.The preliminary study of animals and humans is shown, can alleviate the symptom of some disease, such as diabetes, parkinson, liver failure and congestive heart failure by being implanted in the stem cell of In vitro culture.But, due to many reasons, the ethical issues such as produced with the use of hESC, and our extremely limited knowledge to this field, make slow progress in this field.Due to disadvantages mentioned above, people have begun to use the adult stem cell from human marrow to treat some cancer, such as leukemia (leukemia).But, since it is desired that the surgical operation of intrusive mood, a large amount of expert and specialist center, this therapy can not systematically be applied, and still depended on the wish of clinician.Some researcheres are just developing cell therapy, and this therapy is based on the stem cell being isolatable from umbilical cord.But, the stem cell population that can extract from umbilical cord is the fewest, and this makes this process very long and expensive.Additionally, the stem cell being isolatable from umbilical cord can not be compatible with each recipient immune system, this can cause immunoreation, causes transplant rejection.
Containing multiple stem cell in Placenta Hominis, including the pluripotent stem cell etc. of mescenchymal stem cell, hematopoietic stem cell, endothelial stem cell and unknown function.
Mescenchymal stem cell, English name is mesenchymal stem cells (MSC), also has other title, such as marrow stromal cell, fat stem cell, pluripotency stromal cell etc., refers to that single or a group meets the cell of international uniform standard.Mescenchymal stem cell is present in Various Tissues and organ, including bone marrow, fat, umbilical cord, Placenta Hominis, liver, brain, sclerotin etc., inside and outside is respectively provided with the ability to the differentiation of bone, cartilage and fatty tissue, has the effects such as immunomodulating, hematopoiesis support, angiogenesis promoting.
Hematopoietic stem cell refers to not yet fully-developed cell, it it is the origin of all hematopoietic cells and immunocyte, it is possible not only to be divided into erythrocyte, leukocyte and platelet, it is also possible to cross-system is divided into the cell of various histoorgan, has self renewal Multidirectional Differentiation and potential of going back to the nest.General derived from hematopoietic precursor cells is in three channels: marrow hemopoietic stem cells；Peripheral blood hematopoietic stem cells；Umbilical cord blood hematopoietic stem cell.
Summary of the invention
It is an object of the invention to provide a kind of Placenta Hominis pluripotent stem cell and extract preparation method.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of Placenta Hominis pluripotent stem cell extracting method, comprises the following steps:
(1) pretreatment: obtain placenta tissue under aseptic condition, repeatedly rinse through PBS, remove placental blood, use iodophor disinfection umbilical cord and Placenta Hominis surface；Normal saline fully rinses and remains umbilical cord and Placenta Hominis, at least three times；
(2) Placenta Hominis pluripotent stem cell separation and Culture: placenta tissue is cut into 1 × 1 × 1mm3Fragment, placenta tissue fragment is placed in sterile centrifugation tube, utilizes 0.25% trypsin and 0.2% type Ⅳ collagen enzymic digestion placenta tissue fragment, remove tissue and leave and take Digestive system, it is centrifuged to collect and to obtain cell precipitation, by cell precipitation be resuspended in containing the hyclone α-MEM culture medium that volume fraction is 10%, at 37 DEG C, volume fraction be 5% CO2 gas incubator in cultivate, cultivate the later half amount of 60h and change liquid, removing non-attached cell, within every 5 days, change liquid once, conventional method is frozen；
(3) Immune expression: flow cyctometry analysis: CD73, CD90 and CD105 cell is expressed.
Described step (2) placenta tissue fragment digests 30min at a temperature of 37 DEG C.
Described step (2) centrifugal condition is: 1500r/min, centrifugal 5min.
In described step (2), the volume ratio of 0.25% trypsin and 0.2% type Ⅳ collagenase is 1:1.
Beneficial effects of the present invention: the present invention aseptically obtains Placenta Hominis, then Placenta Hominis is carried out PBS repeatedly rinse, Placenta Hominis surface sterilization, normal saline flushing Placenta Hominis series of preprocessing, ensure that the Placenta Hominis obtained is fresh, it is easy to ensure the activity of pluripotent stem cell in placenta tissue, utilizing 0.25% trypsin and 0.1%I Collagenase Type digestion placenta tissue fragment, the separation efficiency of pluripotent stem cell is high.
Detailed description of the invention
Below in conjunction with the embodiment of the present invention, technical scheme is clearly and completely described, it is clear that described embodiment is only a part of embodiment of the present invention rather than whole embodiments.Based on the embodiment in the present invention, all other embodiments that those of ordinary skill in the art are obtained under not making creative work premise, broadly fall into the scope of protection of the invention.
Embodiment 1
Preparation D-Hanks liquid: in the tri-distilled water of 800ml made fresh, it is sequentially added into sodium chloride 8.00g, potassium chloride 0.40g, disodium hydrogen phosphate 0.06g, potassium dihydrogen phosphate 0.06g, sodium bicarbonate 0.35g, phenol red 0.02g, after being completely dissolved, not enough distilled water is to 1000ml, measures pH7.2-7.4, subpackage 250ml vial, autoclaving 20min, room temperature cools down, 4 DEG C of preservations, and the used time is heated to 37 DEG C.
Preparation trypsin solution: weigh trypsin 250mg, EDTA20mg；Taking 80mlD-Hanks liquid, be completely dissolved by trypsin, add D-Hanks liquid to 100ml, and add EDTA solution and dissolve, its final concentration of 0.25% trypsin-0.02%EDTA solution, with the worry device sucking filtration of 0.1um, degerming to be placed in 4 DEG C of refrigerators standby.
Preparation type Ⅳ collagenase: taking collagenase IV type stock solution 0.2ml, addition D-Hanks liquid is to 100ml, degerming with the filter sucking filtration of 0.1um, is placed in 4 DEG C of refrigerators standby.
A kind of Placenta Hominis pluripotent stem cell extracting method, comprises the following steps:
(1) pretreatment: obtain placenta tissue under aseptic condition, repeatedly rinse through PBS, remove placental blood, use iodophor disinfection umbilical cord and Placenta Hominis surface；Normal saline fully rinses and remains umbilical cord and Placenta Hominis, at least three times；
(2) Placenta Hominis pluripotent stem cell separation and Culture: placenta tissue is cut into 1 × 1 × 1mm3Fragment, placenta tissue fragment is placed in sterile centrifugation tube, utilize 0.25% trypsin and 0.2% type Ⅳ collagen enzymic digestion placenta tissue fragment, the volume ratio of 0.25% trypsin and 0.2% type Ⅳ collagenase is 1:1, 30min is digested at a temperature of 37 DEG C, remove tissue and leave and take Digestive system, it is centrifuged to collect and to obtain cell precipitation, centrifugal condition is: 1500r/min, centrifugal 5min, cell precipitation is resuspended in containing the hyclone α-MEM culture medium that volume fraction is 10%, at 37 DEG C, volume fraction be 5% CO2 gas incubator in cultivate, cultivate the later half amount of 60h and change liquid, remove non-attached cell, within every 5 days, change liquid once, conventional method is frozen；
(3) Immune expression: flow cyctometry analysis: CD73, CD90 and CD105 cell is expressed.
In the description of this specification, the description of reference term " embodiment ", " example ", " concrete example " etc. means that the specific features, structure, material or the feature that combine this embodiment or example description are contained at least one embodiment or the example of the present invention.In this manual, the schematic representation to above-mentioned term is not necessarily referring to identical embodiment or example.And, the specific features of description, structure, material or feature can combine in any one or more embodiments or example in an appropriate manner.
Present invention disclosed above preferred embodiment is only intended to help to illustrate the present invention.Preferred embodiment does not has all of details of detailed descriptionthe, is not intended to the detailed description of the invention that this invention is only described yet.Obviously, according to the content of this specification, can make many modifications and variations.These embodiments are chosen and specifically described to this specification, is to preferably explain the principle of the present invention and actual application, so that skilled artisan can be best understood by and utilize the present invention.The present invention is only limited by claims and four corner thereof and equivalent.
Claims (4)
1. a Placenta Hominis pluripotent stem cell extracting method, it is characterised in that comprise the following steps:
(1) pretreatment: obtain placenta tissue under aseptic condition, repeatedly rinse through PBS, remove placental blood,
Use iodophor disinfection umbilical cord and Placenta Hominis surface；Normal saline fully rinses and remains umbilical cord and Placenta Hominis, at least three times；
(2) Placenta Hominis pluripotent stem cell separation and Culture: placenta tissue is cut into 1 × 1 × 1mm3Fragment, by Placenta Hominis
Fragment of tissue is placed in sterile centrifugation tube, utilizes 0.25% trypsin and 0.2% type Ⅳ collagen enzymic digestion Placenta Hominis
Fragment of tissue, removes tissue and leaves and takes Digestive system, and centrifugal collection to obtain cell precipitation, cell precipitation is resuspended in and contains
Volume fraction is the hyclone α-MEM culture medium of 10%, and at 37 DEG C, volume fraction is the carbon dioxide of 5%
Incubator is cultivated, cultivates the later half amount of 60h and change liquid, remove non-attached cell, within every 5 days, change liquid once, often
Rule method is frozen；
(3) Immune expression: flow cyctometry analysis: CD73, CD90 and CD105 cell is expressed.
A kind of Placenta Hominis pluripotent stem cell extracting method the most according to claim 1, it is characterised in that institute
State step (2) placenta tissue fragment at a temperature of 37 DEG C, digest 30min.
A kind of Placenta Hominis pluripotent stem cell extracting method the most according to claim 1, it is characterised in that institute
Stating step (2) centrifugal condition is: 1500r/min, centrifugal 5min.
A kind of Placenta Hominis pluripotent stem cell extracting method the most according to claim 1, it is characterised in that institute
Stating the volume ratio of 0.25% trypsin and 0.2% type Ⅳ collagenase in step (2) is 1:1.
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| CN201610570529.6A CN105969722A (en) | 2016-07-19 | 2016-07-19 | Extraction and preparation method of placenta pluripotent stem cells |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107744526A (en) * | 2017-10-30 | 2018-03-02 | 南京医科大学 | Dry element of sheep and its preparation method and application |
| CN113736727A (en) * | 2021-08-11 | 2021-12-03 | 李琴 | Method for extracting multiple stem cells of placenta |
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| CN101693884A (en) * | 2009-10-21 | 2010-04-14 | 王沁怡 | Method for separating and extracting stem cells from placenta, umbilical cord or adipose tissue |
| CN104928234A (en) * | 2015-07-07 | 2015-09-23 | 河南中科干细胞基因工程有限公司 | Method for extracting various placenta stem cells |
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2016
- 2016-07-19 CN CN201610570529.6A patent/CN105969722A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101693884A (en) * | 2009-10-21 | 2010-04-14 | 王沁怡 | Method for separating and extracting stem cells from placenta, umbilical cord or adipose tissue |
| CN104928234A (en) * | 2015-07-07 | 2015-09-23 | 河南中科干细胞基因工程有限公司 | Method for extracting various placenta stem cells |
Non-Patent Citations (2)
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| 刘洋 等: "胎盘源间充质干细胞分离提取的新方法", 《中国组织工程研究》 * |
| 徐小霞: "人胎盘绒毛层间充质干细胞体外培养的优化及生物学特性的初步分析", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107744526A (en) * | 2017-10-30 | 2018-03-02 | 南京医科大学 | Dry element of sheep and its preparation method and application |
| CN113736727A (en) * | 2021-08-11 | 2021-12-03 | 李琴 | Method for extracting multiple stem cells of placenta |
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Application publication date: 20160928 |