CN109234229A - Method and digestive enzyme compositions used from placenta blood vessel separating mesenchymal stem cell - Google Patents
Method and digestive enzyme compositions used from placenta blood vessel separating mesenchymal stem cell Download PDFInfo
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- CN109234229A CN109234229A CN201811130277.0A CN201811130277A CN109234229A CN 109234229 A CN109234229 A CN 109234229A CN 201811130277 A CN201811130277 A CN 201811130277A CN 109234229 A CN109234229 A CN 109234229A
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- 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
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2474—Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6489—Metalloendopeptidases (3.4.24)
- C12N9/6491—Matrix metalloproteases [MMP's], e.g. interstitial collagenase (3.4.24.7); Stromelysins (3.4.24.17; 3.2.1.22); Matrilysin (3.4.24.23)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/94—Pancreatin
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- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/11—Exodeoxyribonucleases producing 5'-phosphomonoesters (3.1.11)
- C12Y301/11001—Exodeoxyribonuclease I (3.1.11.1)
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The present invention relates to from placenta blood vessel separating mesenchymal stem cell method and digestive enzyme compositions used.The described method comprises the following steps: placenta disinfects in alcohol；Go out placenta blood vessel from separation of placenta；It is cut into fragment, is cleaned with PBS, residual blood stains are filtered out, obtains placenta vascular tissue；Mixed enzyme solution is added to digest；Digestion is terminated, tissue fluid is collected by filtration；Centrifugation gained cell precipitation is original placenta mesenchyma stem cell (P0 generation), is resuspended with DMEM-F12 basal medium, samples and count the quantity and motility rate of karyocyte；Gained cell is frozen with protection liquid is frozen so as to culture of being recovered before using again, or continues passage and/or gained mescenchymal stem cell carries out cellular identification and/or detection, freezes, builds library etc. operates.The method of the present invention can effectively improve the efficiency from the blood vessel separating mesenchymal stem cell of placenta.
Description
Technical field
The present invention relates to the method for separating stem cell from the blood vessel of placenta, in particular to the compartment from the blood vessel of placenta
The method of mesenchymal stem cells, particularly relate to a kind of digestive enzyme compositions using unique formula of the present invention to from
The method of the blood vessel separating mesenchymal stem cell of placenta.It can effectively be improved using the method for the present invention from the blood vessel of placenta and be separated
The efficiency of mescenchymal stem cell.
Background technique
The mescenchymal stem cell of mescenchymal stem cell (mesenchymal stem cell, MSC) such as mankind be earliest from
It is separated in marrow, it is dry thin from mesoblastic a kind of tissue with multi-lineage potential and self-renewal capacity
Born of the same parents, in vivo under external specified conditions have to osteoblast, cartilage cell, fat cell, endothelial cell, nerve cell,
Ability (the Caplan AI.Mesenchymal stem cells.J of a variety of adult cell differentiation such as myocyte, liver cell
Orthop Res.1991,9:641-650.Pittenger MF,Mackay AM,Beck SC,et al.Multilineage
potential of adult human mesenchymal stem cells.Science.1999；284:143-147).Most
It is new research shows that mescenchymal stem cell has immunological regulation and Hematopoiesis Support affect, and be easy to foreign gene and import expression.
Therefore the mescenchymal stem cell not still seed cell in tissue-engineered bone, cartilage and cardiac muscle building, it is important in gene therapy
Carrier cell, and since mescenchymal stem cell promotes hematopoietic reconstitution and the anti-host response function of inhibition of transplant, in hematopoiesis
It is with a wide range of applications in stem cell transplantation and organ transplant.Mescenchymal stem cell has the characteristic of growth-arrested in vitro,
Using this characteristic, people have succeeded to be divided from the Various Tissues such as liver, kidney, pancreas, muscle, cartilage, skin, peripheral blood
From turning out mescenchymal stem cell.
The mescenchymal stem cell reported at present is mainly derived from marrow, is obtained using density-gradient centrifugation method.Although point
It is easy from method, but the operation that donor takes marrow to need to undergo a comparison painful, and had very during materials and after materials
High infection chance；Since the content of MSC in human bone marrow is extremely rare, every 105~106Only about 1 in a mononuclearcell
It is a, and with the increase at age, quantity, proliferation and the differentiation capability of mescenchymal stem cell are remarkably decreased in marrow, make it
It is restricted in research and application especially clinical application.Placenta originating from embryonic development period extraembryonic mesoderm be by
Matter, blood vessel and trophocyte composition, contain a large amount of mesenchyma ingredient.It is newest research shows that rich in dry thin in placenta
Born of the same parents, be separately cultured out from placenta these multipotential stem cells will be opened up for experimental study and clinical application one it is brand-new and abundant
Source.
Existing method of the stem cell to establish placenta stem-cell library that separate from placenta still has shortcomings, such as pure
Degree is insufficient, and/or quantity is not high, and then shows that these methods are not able to satisfy the expectation of people still.Such as CN101270349A
Entitled " placenta mesenchyma stem cell disclosed in (Chinese Patent Application No. 200810061267.6, publication date September in 2008 24 days)
The invention of separation and amplification in vitro cultural method "；CN101693884A (Chinese Patent Application No. 200910117522.9, it is open
Day on April 14th, 2010) disclosed in entitled " a method of the separating and extracting stem cells from placenta, umbilical cord or adipose tissue "
Invention；It is entitled disclosed in CN102146359A (Chinese Patent Application No. 201110005964.1, publication date August in 2011 10 days)
The invention of " method of primary mesenchymal stem cells and serum-free amplification is extracted from placenta ".In addition, Chinese Patent Application No.
201210044648X discloses a kind of method of separating mesenchymal stem cell from placenta.Purity of these methods in extract
And/or it is remained to be further improved in terms of the rate of recovery.
The one kind of mescenchymal stem cell as adult stem cell, derive from mesoderm growing early stage mesoderm, because have highly self
Update, immunoregulation and multi-lineage potential and be concerned.Mescenchymal stem cell is widely present in the various tissues of whole body, special
It is not marrow, adipose tissue, bleeding of the umbilicus.Mescenchymal stem cell in clinical research is mainly from marrow.Traditional method is at present
Stem cell is obtained from marrow under general anesthesia or intravertebral anesthesia, but is only capable of 100~1000 colony of acquisition in every milliliter of marrow
The mesenchymal stem cell of unit is generated, and have to pass through amplification in vitro to obtain the cell number of clinical needs, this side
Method is not only expensive, increases patient suffering, and need to consume longer treatment time, limits mesenchymal stem cell
Potential applicability in clinical practice.
2003, Mitchell etc. first confirmed that the mescenchymal stem cell extracted from umbilical cord has multi-lineage potential,
Then again there are several scholars to be separated to fibroblast-like cells from umbilical cord Wharton jelly, and confirm its with self-renewing proliferation and it is more
To differentiation potential, it is dry thin (hUC-MSCs) to be named as human umbilical cord mesenchymal.Human umbilical cord mesenchymal is dry carefully to be referred to and is present in umbilical cord
Mescenchymal stem cell, umbilical cord compares other mescenchymal stem cells, human umbilical cord mesenchymal is dry thin to have Asia as childbirth waste
Totipotential differentiation potential obtains simple, abundance, cultivation also than about one times fastly of other stem cells, and growth uniformly, has
Much good characteristics as seed cell, such as proliferation activity is high, immunogenicity is low, without oncogenicity, source is sufficient, no ethics
The advantage of problem etc..The dry carefully potentiality in regenerative medicine and organizational engineering of human umbilical cord mesenchymal, have caused people very
Big interest, compared with the MSCs from marrow, human umbilical cord mesenchymal is dry carefully to have superiority, and people's umbilical cord surface is that amnion is covered with
Skin, wherein include two arteries and a vein, umbilical cord perivascular around mucoid connective tissue (referred to as China's Tong Shi glue,
WJ)。
The source of umbilical cord mesenchymal stem cells includes amnion, amnion lower layer, China's Tong Shi glue, cord vessels, cord vessels week
Enclose tissue and bleeding of the umbilicus.At present clinically it is widely applied be magnificent Tong Shi glue source mescenchymal stem cell.It is filled between people's umbilical cord at present
It is still defective in terms of the research of matter stem cell.It specifically includes that (1) umbilical cord acquisition posterior umbilicus is quiet, arterial blood solidification, increases at umbilical cord
The difficulty of reason, while hypercytosis increases the chance of stem cell pollution；(2) tissue adherent method is that umbilical cord is cut into tiny group
It knits block to be attached directly on culture medium, can get primary cell within general 15 days or so；The shortcomings that this method, is that the period is long；
The easy levitating of tissue block, makes it lose the ability for growing cell, reduces cell quantity, and furthermore cell purity is insufficient；(3) enzyme disappears
Change fado to be used in combination using clostridiopetidase A with trypsase, costly although the period is short, condition is not easy to grasp, when room temperature digests
Between it is long then may damaging cells, the short then liquid of digestion time is sticky, it is difficult to obtain sufficient amount cell by centrifugation, and increase
Animal-based protein causes allergic reaction the security risk with cross-infection in process of clinical application.
In recent years cord vessels week stem cell is considered because of its high proliferation activity, high Clone formation power and multi-lineage potential
It is the progenitor cells of mescenchymal stem cell, there is extensive potential applicability in clinical practice.2013, the researchs such as Tsang WP were pointed out, CD146
+ perivascular cell can be used as the cell origin of osteanagenesis.
CN105695401A (CN201610189133.7) discloses a kind of umbilical artery and vein blood vessel week stem cell
Preparation and store method, are cultivated by cell culture fluid (DMEM low sugar, 10% fetal calf serum, 1% mycillin are dual anti-),
Than the mescenchymal stem cell for the cord vessels Zhou Laiyuan that common paster method obtains higher purity；Digestive ferment is not used, is avoided
Allergic reaction caused by animal-based protein and cross-infection, and non-proenzyme digestion method isolated cord vessels week is dry thin
Born of the same parents are higher than the mescenchymal stem cell CD146 positive rate in magnificent Tong Shi glue source.
It is intended that having the source of new human mesenchymal stem cell, such as isolated mesenchyma is dry from the blood vessel of placenta
Cell, however the prior art there is not yet from the blood vessel of placenta separating mesenchymal stem cell method.Therefore this field still phase
To being capable of the successful separating mesenchymal stem cell from the blood vessel of placenta.
Summary of the invention
Present invention aim to address the existing deficiencies for obtaining placenta mesenchyma stem cell resource, provide a kind of practical, simple
List, efficiently separating mesenchymal stem cell and the method for optionally establishing stem cell bank from the blood vessel of placenta.Meanwhile the present invention
Another object be that the method for the above-mentioned separating mesenchymal stem cell from placenta blood vessel provides a kind of digestive enzyme compositions.This
Inventor has found the digestive enzyme compositions using special operating method and special prescription, cell purity height obtained and/
Or cell recoveries are high.It is accomplished the present invention is based on this discovery.
Therefore, first aspect present invention provides the method for the separating mesenchymal stem cell from the blood vessel of placenta, this method
The following steps are included:
(1) placenta is soaked in 75% alcohol disinfecting 30 seconds, is then rinsed twice with PBS；
(2) placenta blood vessel is separated from the navel baes position of placenta, squeezes blood vessel with surgical forceps to remove blood stains；
(3) scissors for vessels is broken into 1~2mm^3 fragment, is cleaned with PBS, then filter removal residual blood stains with 300 mesh filter screens,
Obtain placenta vascular tissue；
(4) plus mixed enzyme solution digests；
(5) addition serum terminates digestion, then is filtered with 300 mesh filter screens, collects tissue fluid, and PBS washing merges cleaning solution；
(6) centrifugation obtains cell precipitation, is cleaned one time with PBS, is centrifuged, obtains original placenta mesenchyma stem cell (P0
Generation), it is resuspended with DMEM-F12 basal medium, samples and count the quantity and motility rate of karyocyte；Gained cell with freeze protect
Shield liquid freezes to recover culture before using again or gained cell then carries out the operation of below step；
(7) make cell inoculation to T75 culture bottle, add complete medium culture；
(8) it changes the liquid once within every 3 days in incubation, until cell confluency is up to 80% or more, passage obtains P1 for placenta
Mescenchymal stem cell；
And optional following one or more steps:
(9) for placenta mesenchyma stem cell obtained by step (8) carry out cellular identification and/or detection (e.g., including but not
It is limited to, at rouge, skeletonization and at cartilage, flow cytometer detection, HLA identification, cell activity, cell contamination, hereditary disease, HLA-ABC/DR
Distribution type)；
(10) placenta mesenchyma stem cell after passage obtained by step (8) is frozen in liquid nitrogen；
(11) database of the placenta stem-cell comprising information above is established, and makes freezing for the database and step (10)
Cell is associated.
According to method of the first aspect of the present invention, wherein mixed enzyme solution described in step (4) is the PBS for being added to mixed enzyme
Buffer.That is, it is with liquid medium that the mixed enzyme solution, which is with PBS buffer solution, supplement adds corresponding type and disappears with what is measured thereto
Change enzyme, or even can also add other materials on this basis.
According to method of the first aspect of the present invention, wherein include in mixed enzyme solution described in step (4): 0.1~0.3%
Such as 0.2% clostridiopetidase A II, 0.1~0.2% such as 0.15% clostridiopetidase A IV, 0.05~0.15% such as 0.1% deoxyribose core
Sour enzyme I.
According to method of the first aspect of the present invention, wherein add mixed enzyme solution to digest 0.5~2h in step (4), such as digest
1h。
According to method of the first aspect of the present invention, described in step (6) centrifugation be, for example, with 1000~2000rpm for example
1500rpm is centrifuged 3~7min such as 5min.
According to method of the first aspect of the present invention, inoculum density is 0.5~2x10^5/cm^2 in step (7), such as is connect
Kind density is 1x10^5/cm^2.
According to method of the first aspect of the present invention, in step (7), the complete medium is consisting of: DMEM-F12+
15%FBS+10ng/ml basic fibroblast growth factor (BFGF).
According to method of the first aspect of the present invention, in step (7), the culture is in 37 DEG C, 5%CO2In incubator
It is cultivated.
According to method of the first aspect of the present invention, in step (9), the cytoactive detection is to utilize Trypan Blue
Method counts the number for freezing front and back living cells.
According to method of the first aspect of the present invention, in step (9), the cell contamination detection is trained using a small amount of cell
Support, detection cell whether the pollution by fungi and bacterium.In one embodiment, the cell contamination detection is to utilize disease
Former method, detection cell whether by be selected from following one or more infection: Hepatitis B virus, hepatitis, AIDS virus,
Cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM
With EBV-IgA, TRUST.
According to method of the first aspect of the present invention, in step (9), the hereditary disease detection is to utilize molecular genetics
Method, detection freeze-stored cell whether there is hereditary disease.
According to method of the first aspect of the present invention, in step (9), the HLA-ABC/DR distribution type is detection cell HLA-
ABC/DR phenotype.
According to method of the first aspect of the present invention, in step (10), the placenta mesenchyma stem cell is dropped through program
Warm process freezes in liquid nitrogen.
According to method of the first aspect of the present invention, in step (10), the placenta mesenchyma stem cell is present in cell
In frozen stock solution.In one embodiment, which includes 50% low sugar DMEM culture solution, 40%FBS, 10% diformazan
Base sulfoxide.
According to method of the first aspect of the present invention, the cell in step (11), in the database including and being saved
All relevant data, including but not limited to: the biological characteristics testing result of cell, multi-lineage potential qualification result, thin
Extracellular molecule genetic diagnosis result, fetus and its particulars of parent.
In addition, obtaining a kind of placenta mesenchyma isolated from placenta blood vessel in first aspect present invention method
Stem cell.Therefore second aspect of the present invention provides a kind of placenta mesenchyma stem cell isolated from placenta blood vessel.
Placenta mesenchyma stem cell according to a second aspect of the present invention is any embodiment party according to a first aspect of the present invention
What case the method obtained.
Placenta mesenchyma stem cell according to a second aspect of the present invention, cell purity are greater than 90%.In an embodiment party
In case, for the placenta mesenchyma stem cell after 1 more than generation passes on, cell purity is greater than 90%.
Further, third aspect present invention provides a kind of method from placenta blood vessel isolation of human placenta mesenchymal stem
Used in digestive enzyme compositions, which is the PBS buffer solution containing tissue digestion enzyme, this contains tissue digestion
The PBS buffer solution of enzyme is that addition is selected from following one or more digestive ferments: dispase, pancreatin, deoxidation in PBS buffer solution
Ribalgilase I (DNase I), clostridiopetidase A II, clostridiopetidase A IV, hyaluronidase.In one embodiment, the digestive ferment group
Closing includes following digestive ferment: deoxyribonuclease I (DNase I), clostridiopetidase A II, clostridiopetidase A IV in object.
Digestive enzyme compositions according to a third aspect of the present invention, the digestive enzyme compositions are the PBS containing the digestive ferment
Buffer.In one embodiment, include in the PBS buffer solution containing the digestive ferment: 0.1~0.3% such as 0.2% glue
Such as such as 0.1% deoxyribonuclease I of 0.15% clostridiopetidase A IV, 0.05~0.15% of protoenzyme II, 0.1~0.2%.
Digestive enzyme compositions according to a third aspect of the present invention, the digestive enzyme compositions are the PBS containing the digestive ferment
Buffer.In one embodiment, include in the PBS buffer solution containing the digestive ferment: 0.2% clostridiopetidase A II, 0.15%
Clostridiopetidase A IV, 0.1% deoxyribonuclease I.
Digestive enzyme compositions according to a third aspect of the present invention, wherein described dry thin from placenta blood vessel separation placenta mesenchyma
The method of born of the same parents comprising following steps:
(1) placenta is soaked in 75% alcohol disinfecting 30 seconds, is then rinsed twice with PBS；
(2) placenta blood vessel is separated from the navel baes position of placenta, squeezes blood vessel with surgical forceps to remove blood stains；
(3) scissors for vessels is broken into 1~2mm^3 fragment, is cleaned with PBS, then filter removal residual blood stains with 300 mesh filter screens,
Obtain placenta vascular tissue；
(4) plus mixed enzyme solution digests；
(5) addition serum terminates digestion, then is filtered with 300 mesh filter screens, collects tissue fluid, and PBS washing merges cleaning solution；
(6) centrifugation obtains cell precipitation, is cleaned one time with PBS, is centrifuged, obtains original placenta mesenchyma stem cell (P0
Generation), it is resuspended with DMEM-F12 basal medium, samples and count the quantity and motility rate of karyocyte；Gained cell with freeze protect
Shield liquid freezes to recover culture before using again or gained cell then carries out the operation of below step；
(7) make cell inoculation to T75 culture bottle, add complete medium culture；
(8) it changes the liquid once within every 3 days in incubation, until cell confluency is up to 80% or more, passage obtains P1 for placenta
Mescenchymal stem cell；
And optional following one or more steps:
(9) for placenta mesenchyma stem cell obtained by step (8) carry out cellular identification and/or detection (e.g., including but not
It is limited to, at rouge, skeletonization and at cartilage, flow cytometer detection, HLA identification, cell activity, cell contamination, hereditary disease, HLA-ABC/DR
Distribution type)；
(10) placenta mesenchyma stem cell after passage obtained by step (8) is frozen in liquid nitrogen；
(11) database of the placenta stem-cell comprising information above is established, and makes freezing for the database and step (10)
Cell is associated.
Digestive enzyme compositions according to a third aspect of the present invention, wherein mixed enzyme solution described in step (4) be added to it is mixed
The PBS buffer solution of synthase.
Digestive enzyme compositions according to a third aspect of the present invention wherein include: 0.1 in mixed enzyme solution described in step (4)
~0.3% such as 0.2% clostridiopetidase A II, 0.1~0.2% such as 0.15% clostridiopetidase A IV, 0.05~0.15% such as 0.1% take off
Oxygen ribalgilase I.
Digestive enzyme compositions according to a third aspect of the present invention wherein add mixed enzyme solution to digest 0.5~2h in step (4),
Such as digestion 1h.
Digestive enzyme compositions according to a third aspect of the present invention, described in step (6) centrifugation be, for example, with 1500rpm from
Heart 5min.
Digestive enzyme compositions according to a third aspect of the present invention, inoculum density is 0.5~2x10^5/cm^ in step (7)
2, such as inoculum density is 1x10^5/cm^2.
Digestive enzyme compositions according to a third aspect of the present invention, in step (7), the complete medium consisting of:
DMEM-F12+15%FBS+10ng/ml basic fibroblast growth factor (BFGF).
Digestive enzyme compositions according to a third aspect of the present invention, in step (7), the culture is in 37 DEG C, 5%CO2
It is cultivated in incubator.
Digestive enzyme compositions according to a third aspect of the present invention, in step (9), the cytoactive detection is to utilize platform
Expect that blue decoration method counts the number for freezing front and back living cells.
Digestive enzyme compositions according to a third aspect of the present invention, in step (9), the cell contamination detection is using few
Measure cell culture, detection cell whether the pollution by fungi and bacterium.In one embodiment, the cell contamination detection
Using aetology method, whether detection cell by being selected from following one or more infection: Hepatitis B virus, hepatitis,
AIDS virus, cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/
2Ab, CMV-IgM and EBV-IgA, TRUST.
Digestive enzyme compositions according to a third aspect of the present invention, in step (9), the hereditary disease detection is to utilize molecule
The method of science of heredity, detection freeze-stored cell whether there is hereditary disease.
Digestive enzyme compositions according to a third aspect of the present invention, in step (9), the HLA-ABC/DR distribution type is detection
Cell HLA-ABC/DR phenotype.
Digestive enzyme compositions according to a third aspect of the present invention, in step (10), the placenta mesenchyma stem cell is
It is frozen in liquid nitrogen through program temperature-fall period.
Digestive enzyme compositions according to a third aspect of the present invention, in step (10), the placenta mesenchyma stem cell is deposited
It is in cells frozen storing liquid.In one embodiment, the cells frozen storing liquid include 50% low sugar DMEM culture solution, 40%FBS,
10% dimethyl sulfoxide.
Digestive enzyme compositions according to a third aspect of the present invention include and are protected in step (11) in the database
All relevant data of the cell deposited, including but not limited to: the biological characteristics testing result of cell, multi-lineage potential identification
As a result, cellular elements genetic diagnosis result, fetus and its particulars of parent.
The present invention is further illustrated below.Cited text in document cited in the present invention and the document
It offers, their full content is incorporated herein by reference.
In the present invention, in any technical solution of either side of the present invention, any technical characteristic is equally applicable to this
Any embodiment in either invention face, as long as they will not cause contradiction, and this be mutually applicable in if necessary may be used
To be suitably modified.
In the present invention, term " placenta mesenchyma stem cell " refers to the mescenchymal stem cell from placenta.Therefore exist
In the present invention, more particularly in context of the invention, term " placenta mesenchyma stem cell " can with " placenta stem-cell ",
" stem cell ", " mescenchymal stem cell " are used interchangeably, unless otherwise clearly indicating.Moreover, it relates to method be from tire
The separation of disk blood vessel obtains placenta mesenchyma stem cell, therefore more specifically, placenta mesenchyma stem cell of the present invention refers to source
In the placenta mesenchyma stem cell of placenta blood vessel.
In the present invention, term " PBS buffer solution " or " PBS " refer to phosphate buffer.Those skilled in the art are ripe
Know the general formula and preparation method and their general aspects such as pH value or pH of the PBS used under situation of the present invention
Range, and these PBS buffer solution are usually the pre-mixing liquor (or prewired powder) that can be obtained through commercial channels, such as this
The PBS of invention field is usually the commercialization buffer of pH7.4 (± 0.1), such as the PBS buffer solution of HyClone brand；Ability
It include 137mM sodium chloride, 2.7nM potassium chloride and 10mM phosphate radical in PBS buffer solution composition when the application of domain classics, in this hair
In bright if not otherwise specified, PBS used using when composition be the composition.
In the present invention, term " placenta " refers to newborn fetal placenta, particularly relates to the placenta within 4 hours postpartum.
The method of either side according to the present invention, wherein also adding 0.02~0.05% sodium glutamate in the mixed enzyme solution
Include in 0.05~0.1% sodium alginate, such as the mixed enzyme solution: 0.1~0.3% such as 0.2% clostridiopetidase A II, 0.1
~0.2% such as such as 0.1% deoxyribonuclease I of 0.15% clostridiopetidase A IV, 0.05~0.15%, 0.02~0.05%
Such as 0.03% sodium glutamate and 0.05~0.1% such as 0.075% sodium alginate.Such as containing about in the mixed enzyme solution
0.02%, about 0.03%, about 0.04% or about 0.05% sodium glutamate and about 0.05%, about 0.06%, about 0.075%,
Or about 0.1% sodium alginate.It has been had now surprisingly been found that, added simultaneously into the PBS buffer solution containing tissue digestion enzyme
A small amount of sodium glutamate and sodium alginate can significantly improve cell yield, this will have meaning of crucial importance.This raising
The test of cell yield is achieved as follows.1~embodiment of embodiment 3 as follows, wherein the placenta blood vessel of step (3) amount shown
Tissue obtains the karyocyte of quantity shown in step (6), and every 1 gram of placenta vascular tissue can harvest 1.93~2.10 × 10^7
Karyocyte, this data, for the yield of cell, can reflect the MSC preparation efficiency of the operating process before step (6) as P0；
The present inventor has attempted in 1 method and step of the embodiment of the present invention (4) using digestive ferment formula disclosed in several prior arts
Digestive ferment of the present invention is replaced, as a result P0 is respectively less than 0.1 × 10^7 karyocyte/g placenta vascular tissue for cell yield, remote low
In yield of the present invention, digestive ferment used in the embodiment 1 for example, by using Chinese Patent Application No. 201710454588.1 (contains
0.1mg/mL dispase, 0.25mg/mL pancreatin, 0.25mg/mL DNase I, 1mg/mL clostridiopetidase A IV, 1mg/mL hyalomitome
The PBS buffer solution of sour enzyme) P0 be respectively less than 0.087 × 10^7 karyocyte/g placenta vascular tissue for cell yield.Although
The above-mentioned mixture slaking enzyme of the present invention has been obtained for the yield being quite satisfied with, however those skilled in the art still expect to improve this
Kind yield.It is supplemented in embodiment A in a supplement example of the invention, the method for respectively referring to 1~embodiment of embodiment 3,
Different is only supplement 0.03% sodium glutamate of addition and 0.075% sodium alginate in mixed enzyme solution, the results show that three benefits
The karyocyte yield for enriching step (1) to step (6) in example is respectively 9.26 × 10^7 karyocyte/g placenta blood vessel group
It knits, 9.57 × 10^7 karyocyte/g placenta vascular tissue, 9.36 × 10^7 karyocyte/g placenta vascular tissue, shows
P0 can be significantly improved for the yield of cell after supplement addition sodium glutamate and sodium alginate in mixed enzyme solution.Of the invention one
A supplement example is supplemented in embodiment B, and the method for respectively referring to embodiment 1, different is only to supplement to add in mixed enzyme solution
Add 0.02% sodium glutamate/0.075% sodium alginate combination or supplement addition 0.03% sodium glutamate/0.1% sodium alginate
Combination or supplement addition 0.04% sodium glutamate/0.05% sodium alginate combination or supplement 0.05% glutamic acid of addition
Sodium/0.06% sodium alginate combined result shows that the karyocyte yield of step (1) to step (6) divides in four supplement examples
Not Wei 9.33 × 10^7 karyocyte/g placenta vascular tissue, 10.37 × 10^7 karyocyte/g placenta vascular tissue,
10.02 × 10^7 karyocyte/g placenta vascular tissue, 9.87 × 10^7 karyocyte/g placenta vascular tissue, show
P0 can be significantly improved for the yield of cell after supplement addition sodium glutamate and sodium alginate in mixed enzyme solution.At of the invention one
It supplements example to supplement in embodiment C, the method for respectively referring to embodiment 1, different is only to supplement addition in mixed enzyme solution
0.03% sodium glutamate or supplement addition 0.075% sodium alginate or supplement addition 0.03% sodium glutamate and
0.075% mannitol, the results show that the karyocyte yield of step (1) to step (6) is respectively 1.86 in three supplement examples
× 10^7 karyocyte/g placenta vascular tissue, 1.94 × 10^7 karyocyte/g placenta vascular tissue, 1.73 × 10^7
A karyocyte/g placenta vascular tissue shows only to add a kind of above-mentioned enriching substance or use mannitol instead that P0 cannot be effectively improved
For the yield of cell.
The invention discloses a kind of in the method for high yield separating mesenchymal stem cell from placenta blood vessel, and utilizes this
Method saves placenta mesenchyma stem cell and establishes placenta stem-cell library.The present inventor is between summarizing previous be separately cultured
It is successful from placenta in conjunction with stationary culture using Various Tissues digestive ferment mixture slaking tissue block on the basis of mesenchymal stem cells
Blood vessel in isolated a large amount of mescenchymal stem cells.The mescenchymal stem cell that the method for the present invention obtains is with high purity, quantity is more, tool
Have biological characteristics identical with mesenchymal stem cell, can to osteoblast, cartilage cell, fat cell, endothelial cell,
The differentiation such as nerve cell.Due in placenta stem cell compared with adult stem cell naivety, rich content, clinically have widely answer
With prospect, we will derive from the placenta mesenchyma stem cell of placental blood pipe as bleeding of the umbilicus with conventional cell freezing method
It freezes, establishes placenta stem-cell library, lay the foundation for the further investigation and clinical treatment of later stem cell.
Due to candidate stem cell rich in bleeding of the umbilicus, people establish unbilical blood bank, and umbilical hemopoietic stem cell, this is important
Living resources store, provide a kind for the treatment of means for a variety of diseases in the blood system and disease of immune system.Same placenta
As a kind of more importantly stem cell resource, we are freezed mescenchymal stem cell with conventional cell freezing method
It is saved for a long time in -196 degrees Celsius of profound hypothermia liquid nitrogen, establishes placenta stem-cell library, save kind for stem-cell therapy in the future
Son.
Practical separate that obtain mesenchyma dry with high yield simply from placenta blood vessel the object of the present invention is to provide a kind of
Cell and the method for establishing placenta stem-cell library include the following steps: that (1) within four hours postpartum, aseptically will
Placenta is soaked in 75% alcohol disinfecting 30 seconds, is then rinsed twice with PBS；(2) placental blood is separated from the navel baes position of placenta
Pipe squeezes blood vessel with surgical forceps to remove blood stains；(3) scissors for vessels is broken into about 1.5mm^3 fragment, is cleaned with PBS, then with 300
Mesh filter screen filtering removal residual blood stains, obtain placenta vascular tissue；(4) plus mixed enzyme solution (wherein include: 0.2% clostridiopetidase A II,
0.15% clostridiopetidase A IV, 0.1% deoxyribonuclease I, in PBS buffer solution) digestion 1h；(5) addition serum terminates digestion,
It is filtered again with 300 mesh filter screens, collects tissue fluid, PBS washing merges cleaning solution；(6) centrifugation (being centrifuged 5min with 1500rpm) obtains
It to cell precipitation, is cleaned one time with PBS, centrifugation (is centrifuged 5min with 1500rpm), obtains original placenta mesenchyma stem cell
(P0 generation) is resuspended with DMEM-F12 basal medium, samples and count the quantity and motility rate of karyocyte；Gained cell is with freezing
Protection liquid freezes to recover culture before using again or gained cell then carries out the operation of below step；(7) connect cell
Kind adds complete medium (consisting of: DMEM-F12+15%FBS+10ng/ml basic fibroblast to T75 culture bottle
Growth factor (BFGF)) culture (37 DEG C, 5%CO2Incubator)；(8) it changes the liquid once within every 3 days in incubation, until cell melts
For conjunction rate up to 80% or more, passage obtains P1 for placenta mesenchyma stem cell.
Practical separate that obtain mesenchyma dry with high yield simply from placenta blood vessel the object of the present invention is to provide a kind of
Cell and the method for establishing placenta stem-cell library include the following steps: that (1) within four hours postpartum, aseptically will
Placenta is soaked in 75% alcohol disinfecting 30 seconds, is then rinsed twice with PBS；(2) placental blood is separated from the navel baes position of placenta
Pipe squeezes blood vessel with surgical forceps to remove blood stains；(3) scissors for vessels is broken into 1mm^3 fragment, is cleaned with PBS, then filtered with 300 mesh
Net filtration removal residual blood stains, obtain placenta vascular tissue；(4) plus mixed enzyme solution (wherein include: 0.3% clostridiopetidase A II,
0.1% clostridiopetidase A IV, 0.15% deoxyribonuclease I, in PBS buffer solution) digestion 0.5h；(5) termination of addition serum disappears
Change, then filtered with 300 mesh filter screens, collect tissue fluid, PBS washing merges cleaning solution；(6) centrifugation (7min is centrifuged with 1000rpm)
Cell precipitation is obtained, is cleaned one time with PBS, centrifugation (is centrifuged 7min with 1000rpm), obtains original placenta mesenchyma stem cell
(P0 generation) is resuspended with DMEM-F12 basal medium, samples and count the quantity and motility rate of karyocyte；Gained cell is with freezing
Protection liquid freezes to recover culture before using again or gained cell then carries out the operation of below step；(7) connect cell
Kind adds complete medium (consisting of: DMEM-F12+15% to T75 culture bottle (inoculum density are as follows: 2*10^5/cm^2)
FBS+10ng/ml basic fibroblast growth factor (BFGF)) culture (37 DEG C, 5%CO2Incubator)；(8) in incubation
It changes the liquid once within every 3 days, until cell confluency is up to 80% or more, passage obtains P1 for placenta mesenchyma stem cell.
Practical separate that obtain mesenchyma dry with high yield simply from placenta blood vessel the object of the present invention is to provide a kind of
Cell and the method for establishing placenta stem-cell library include the following steps: that (1) within four hours postpartum, aseptically will
Placenta is soaked in 75% alcohol disinfecting 30 seconds, is then rinsed twice with PBS；(2) placental blood is separated from the navel baes position of placenta
Pipe squeezes blood vessel with surgical forceps to remove blood stains；(3) scissors for vessels is broken into 2mm^3 fragment, is cleaned with PBS, then filtered with 300 mesh
Net filtration removal residual blood stains, obtain placenta vascular tissue；(4) plus mixed enzyme solution (wherein include: 0.1% clostridiopetidase A II,
0.2% clostridiopetidase A IV, 0.05% deoxyribonuclease I, in PBS buffer solution) digestion 2h；(5) addition serum terminates digestion,
It is filtered again with 300 mesh filter screens, collects tissue fluid, PBS washing merges cleaning solution；(6) centrifugation (being centrifuged 3min with 2000rpm) obtains
It to cell precipitation, is cleaned one time with PBS, centrifugation (is centrifuged 3min with 2000rpm), obtains original placenta mesenchyma stem cell
(P0 generation) is resuspended with DMEM-F12 basal medium, samples and count the quantity and motility rate of karyocyte；Gained cell is with freezing
Protection liquid freezes to recover culture before using again or gained cell then carries out the operation of below step；(7) connect cell
Kind adds complete medium (consisting of: DMEM-F12+15%FBS+10ng/ml basic fibroblast to T75 culture bottle
Growth factor (BFGF)) culture (37 DEG C, 5%CO2Incubator)；(8) it changes the liquid once within every 3 days in incubation, until cell melts
For conjunction rate up to 80% or more, passage obtains P1 for placenta mesenchyma stem cell.
Operation of the present invention is simple, convenient and practical, can obtain from the separation of placenta blood vessel and obtain a large amount of placenta mesenchyma stem cell,
These placenta mesenchyma stem cells differentiation performance is good, has to osteoblast, fat cell, cartilage cell, endothelial cell, nerve
The ability of the cell differentiations such as cell.Compared with existing method: MSC mainly uses modus operandi to extract donor bone marrow or perfusion at present
Method separates placenta, and adhere-wall culture obtains.The method gets that cell quantity is few, and donor has in taking marrow and infection after taking marrow
It may.Present invention success separation from placenta blood vessel obtains a large amount of higher mescenchymal stem cells of purity, and establishes with this method
The stem cell of this great application prospect is laid in placenta stem-cell library.The method is simple and easy to do, and since placenta is as bleeding of the umbilicus,
Cell Component is inmatureer, from a wealth of sources, is conveniently easy to get, therefore method of the invention will have extensively in the clinical application of stem cell
General prospect.
Detailed description of the invention
A, B, C, D, E of Fig. 1 is flow cytometry identification of M SC surface marker result；Wherein, as shown in figure B, D, E,
CD73, CD90, CD105 positive rate are all larger than 98%, and as shown in figure C, CD11b, CD34, CD45, CD19, HLA-DR positive rate are equal
Less than 2%.
Specific embodiment
The present invention can be further described by the following examples, however, the scope of the present invention and unlimited
In following embodiments.One of skill in the art, can be with it is understood that under the premise of without departing substantially from the spirit and scope of the present invention
Various change and modification are carried out to the present invention.The present invention carries out the material and test method arrived used in test general
And/or specific description.Although to realize the present invention many materials and operating method used in purpose be it is known in the art that
But the present invention still makees description as detailed as possible herein.
Embodiment 1, secondary culture, freezes the separation of placenta MSC
(1) within four hours postpartum, placenta is aseptically soaked in 75% alcohol disinfecting 30 seconds, is then used
PBS is rinsed twice；
(2) placenta blood vessel is separated from the navel baes position of placenta, squeezes blood vessel with surgical forceps to remove blood stains；
(3) scissors for vessels is broken into about 1.5mm^3 fragment, is cleaned with PBS, then filter removal residual blood stains with 300 mesh filter screens,
Obtain placenta vascular tissue (one placenta of the present embodiment obtains 5.8g placenta vascular tissue)；
(4) plus mixed enzyme solution (wherein includes: 0.2% clostridiopetidase A II, 0.15% clostridiopetidase A IV, 0.1% DNA
Enzyme I, in PBS buffer solution) digestion 1h；
(5) addition serum terminates digestion, then is filtered with 300 mesh filter screens, collects tissue fluid, and PBS washing merges cleaning solution；
(6) centrifugation (with 1500rpm be centrifuged 5min) obtain cell precipitation, clean one time with PBS, be centrifuged (with 1500rpm from
Heart 5min), original placenta mesenchyma stem cell (P0 generation) is obtained, is resuspended with DMEM-F12 basal medium, is sampled and count
The quantity and motility rate (obtaining the number of nucleated cells of 1.22 × 10^8 or so, activity is 90% or more) of karyocyte；Gained cell
It is frozen with protection liquid is frozen to recover culture before using again or gained cell then carries out the operation of below step；
(7) make cell inoculation to T75 culture bottle (inoculum density are as follows: 1*10^5/cm^2), add complete medium (its group
Become: DMEM-F12+15%FBS+10ng/ml basic fibroblast growth factor (BFGF)) culture (37 DEG C, 5%CO2Training
Support case)；
(8) it changes the liquid once within every 3 days in incubation, until (usual 10 days or so i.e. up to 80% or more for cell confluency
Can), passage, obtaining P1, (thus harvest is up to the mescenchymal stem cell of 2.54 × 10^8 quantity, living for placenta mesenchyma stem cell
Property is up to 95% or more).
Then as the method for step (8) continues the generation for being passaged to needs.Gained MSC carries out the survey of subsequent experimental example
It is fixed.
Embodiment 2, secondary culture, freezes the separation of placenta MSC
(1) within four hours postpartum, placenta is aseptically soaked in 75% alcohol disinfecting 30 seconds, is then used
PBS is rinsed twice；
(2) placenta blood vessel is separated from the navel baes position of placenta, squeezes blood vessel with surgical forceps to remove blood stains；
(3) scissors for vessels is broken into 1mm^3 fragment, is cleaned with PBS, then filter removal residual blood stains with 300 mesh filter screens, obtained
Placenta vascular tissue (one placenta of the present embodiment obtains 5.5g placenta vascular tissue)；
(4) plus mixed enzyme solution (wherein includes: 0.3% clostridiopetidase A II, 0.1% clostridiopetidase A IV, 0.15% DNA
Enzyme I, in PBS buffer solution) digestion 0.5h；
(5) addition serum terminates digestion, then is filtered with 300 mesh filter screens, collects tissue fluid, and PBS washing merges cleaning solution；
(6) centrifugation (with 1000rpm be centrifuged 7min) obtain cell precipitation, clean one time with PBS, be centrifuged (with 1000rpm from
Heart 7min), original placenta mesenchyma stem cell (P0 generation) is obtained, is resuspended with DMEM-F12 basal medium, is sampled and count
The quantity and motility rate (obtaining the number of nucleated cells of 1.06 × 10^8 or so, activity is 90% or more) of karyocyte；Gained cell
It is frozen with protection liquid is frozen to recover culture before using again or gained cell then carries out the operation of below step；
(7) make cell inoculation to T75 culture bottle (inoculum density are as follows: 2*10^5/cm^2), add complete medium (its group
Become: DMEM-F12+15%FBS+10ng/ml basic fibroblast growth factor (BFGF)) culture (37 DEG C, 5%CO2Training
Support case)；
(8) it changes the liquid once within every 3 days in incubation, until (usual 10 days or so i.e. up to 80% or more for cell confluency
Can), passage, obtaining P1, (thus harvest is up to the mescenchymal stem cell of 2.18 × 10^8 quantity, living for placenta mesenchyma stem cell
Property is up to 95% or more).
Then as the method for step (8) continues the generation for being passaged to needs.Gained MSC carries out the survey of subsequent experimental example
It is fixed.
Embodiment 3, secondary culture, freezes the separation of placenta MSC
(1) within four hours postpartum, placenta is aseptically soaked in 75% alcohol disinfecting 30 seconds, is then used
PBS is rinsed twice；
(2) placenta blood vessel is separated from the navel baes position of placenta, squeezes blood vessel with surgical forceps to remove blood stains；
(3) scissors for vessels is broken into 2mm^3 fragment, is cleaned with PBS, then filter removal residual blood stains with 300 mesh filter screens, obtained
Placenta vascular tissue (one placenta of the present embodiment obtains 4.7g placenta vascular tissue)；
(4) plus mixed enzyme solution (wherein includes: 0.1% clostridiopetidase A II, 0.2% clostridiopetidase A IV, 0.05% DNA
Enzyme I, in PBS buffer solution) digestion 2h；
(5) addition serum terminates digestion, then is filtered with 300 mesh filter screens, collects tissue fluid, and PBS washing merges cleaning solution；
(6) centrifugation (with 2000rpm be centrifuged 3min) obtain cell precipitation, clean one time with PBS, be centrifuged (with 2000rpm from
Heart 3min), original placenta mesenchyma stem cell (P0 generation) is obtained, is resuspended with DMEM-F12 basal medium, is sampled and count
The quantity and motility rate (obtaining the number of nucleated cells of 0.93 × 10^8 or so, activity is 90% or more) of karyocyte；Gained cell
It is frozen with protection liquid is frozen to recover culture before using again or gained cell then carries out the operation of below step；
(7) make cell inoculation to T75 culture bottle (inoculum density are as follows: 0.5*10^5/cm^2), add complete medium (its
Composition are as follows: DMEM-F12+15%FBS+10ng/ml basic fibroblast growth factor (BFGF)) culture (37 DEG C, 5%CO2
Incubator)；
(8) it changes the liquid once within every 3 days in incubation, until (usual 10 days or so i.e. up to 80% or more for cell confluency
Can), passage, obtaining P1, (thus harvest is up to the mescenchymal stem cell of 1.83 × 10^8 quantity, living for placenta mesenchyma stem cell
Property is up to 95% or more).
Then as the method for step (8) continues the generation for being passaged to needs.Gained MSC carries out the survey of subsequent experimental example
It is fixed.
The Identification of Biological Characteristics of test example 1, placenta MSC
It, can be with by the operation of embodiment 1:
For placenta mesenchyma stem cell obtained by step (8) carry out cellular identification and/or detection (e.g., including but it is unlimited
In matching at rouge, skeletonization and at cartilage, flow cytometer detection, HLA identification, cell activity, cell contamination, hereditary disease, HLA-ABC/DR
Type)；
Placenta mesenchyma stem cell after passage obtained by step (8) is frozen in liquid nitrogen；And/or
The database of the placenta stem-cell comprising information above is established, and is associated the database with freeze-stored cell.
1, cell growth and its Morphological Characteristics
By being separately cultured for embodiment 1, the culture of placenta mononuclearcell can obviously be seen under the microscope after 72 hours
Shuttle shape attached cell will form turbine-like cell clone for 10 days or so, will form the adherent of 80% or so fusion after had digestive transfer culture
Layer.In incubation, it is found that this cellular morphology is relatively uniform, growth rate is fast, and adherent speed is fast, is easily digested by pancreatin, passes
In generation, form and growth characteristic were also without substantially changeing to 15 more than generation.
2, flow cytometry identification of M SC surface marker
By being separately cultured for embodiment 1, the 0th, 1,3,6 generation cells, Flow cytometry cell surface mark are taken respectively
Will, the variation of cell surface marker in dynamic observation incubation.Cell is collected in digestion, takes 8 × 10 after counting6A cell, point
Fill 16 pipes；PBS is washed once, and 1500rpm is centrifuged 10min；Supernatant is abandoned, 100~200 μ l are remained, piping and druming mixes cell；PE mark is added
The CD45 of CD14, CD29, CD31, CD34, CD44, CD54, CD73, CD80, CD86, CD166 antibody and the FITC label of note,
Each 10 μ l of CD105, HLA-ABC, HLA-DR, UEA-1 antibody, and a pipe is set as blank control；At 4 DEG C, it is protected from light
30min；PBS is washed once, and 1500rpm is centrifuged 10min；The cell directly marked abandons supernatant, and the PBS that 200 μ l are added, which is blown and beaten, to be mixed
1% paraformaldehyde of cell, 200 μ l is fixed, and 4 DEG C of to be measured, flow cytometer detections in 3 days are set.
The surface marker of flow cytomery cell, dynamic observation the 0th, 1,3, the cell in 6 generations, without substantially changeing.Stream
Formula testing result shows that CD73, CD90, CD105 positive rate are all larger than 98%, and CD11b, CD34, CD45, CD19, HLA-DR are positive
Rate is respectively less than 2%, concrete outcome such as Fig. 1.
3, the cell cycle of Flow cytometry placenta MSC
By being separately cultured for embodiment 1, cell is long to when 80% or so fusion, and cell about 1 × 10 is collected in digestion6It is a,
PBS, which is washed, to be once added 70% ethyl alcohol and fixes, and 4 DEG C to be measured.When detection, first ethyl alcohol is removed in centrifugation, then is washed once with PBS, is added
RNase I 500u, 37 DEG C of reaction 30min, PBS are washed once, propidium iodide (PI, 50 μ g/ml of final concentration) 1ml are added, room temperature is kept away
Light reaction 20min, upper machine testing cell DNA content.The cell of in vitro culture has typical stem cells hyperplasia special as the result is shown
Point, i.e., only a few cell is in active proliferation period (<1.5%), and most cell is in quiescent stage (>95%).
4, the drafting of placenta MSC growth curve and the measurement of logarithmic growth phase doubling time
By being separately cultured for embodiment 1, logarithmic growth phase cell, digestion is counted, and is trained with the LG-DMEM of 10%FBS
Cell suspension (2 × 10 is made in feeding base4/ ml), every hole is inoculated with 0.5ml in 24 orifice plates, and 37 DEG C, 5%CO2, cultivate under saturated humidity.
3 multiple holes are taken daily, living cell counting number after Trypan Blue calculates average value, is observed continuously 7 days.Using incubation time as horizontal axis,
Cell number is the longitudinal axis, draws cell growth curve.Cell is calculated in the doubling time of logarithmic growth phase with Patterson formula,
That is Td=Tlg2/Lg (Nt/No), Td: the doubling time (h), T: cell increases to the time (h) used in Nt as No, N: cell number.It is logical
The result for crossing daily cell count draws cell growth curve, calculates the doubling time.The cell it can be seen from cell growth curve
Exponential phase of growth was at the 2-4 days.
5, the identification of placenta MSC multi-lineage potential
(1) osteogenic induction
By being separately cultured for embodiment 1, the 3 generations above MSC, by 1 × 105/ hole is inoculated with six orifice plates, is put in 37 DEG C, 5%
CO2, under saturated humidity, after being cultivated for 24 hours in MSC culture medium, use instead and screened the DMEM-HG of FBS containing 10% and fill in rice with being added
Loose 0.1 μM, 50 μM of ascorbyl phosphate, β-phosphoglycerol 10mM, are put in 37 DEG C, 5%CO2, cultivate under saturated humidity, often
3 days half amounts change liquid, and coinduction 2-4 weeks.Alkaline phosphatase staining identification osteoblast is formed, Von Kossa dyeing identification bone knot
Section is formed.
, through screening the DMEM-HG of FBS, 0.1 μM of dexamethasone, 50 μM of ascorbyl phosphate, β-phosphorus is being added containing 10%
Acid glycerol 10mM is cultivated 1 week, and apparent change occurs for cellular morphology, becomes polygonal, class from fusiform fibroblast sample
It is similar to neuronal cell sample, it is prominent that long filiform occurs in cell periphery, and can extend to surrounding.After continuing culture 2 weeks or more, cell
Occurs calcified plaque in matrix, mineralizer gradually appears, and initially forms the small junction structure of multilayer, until after culture 4 weeks, it is seen that obvious
Calcium scoring.Alkaline phosphatase staining is in strong positive reaction at 2 weeks, reaches 95% or more, and the control group not induced is then
Most of is feminine gender, is shown as weakly positive only less than 5%, shows that cell is converted to osteoblast.Von Kossa dyeing can
The calcium deposited in bone tubercle is dyed into black, the visible a large amount of black bone tubercle of induction group has apparent stereochemical structure, and compares
Group is at any time all without positive reaction.
(2) Adipogenic induction
By being separately cultured for embodiment 1, the 3 generations above MSC, by 1 × 105/ hole is inoculated in six orifice plates, is put in 37 DEG C, 5%
CO2, under saturated humidity, after being cultivated for 24 hours in MSC culture medium, use instead and screened the DMEM in high glucose of FBS containing 10%, and be added ground
1 μM of Sai meter Song, 60 μM of indocin, IBMX 0.5mM, 5 μ g/ml of insulin, are put in 37 DEG C, 5%CO2, it cultivates under saturated humidity,
Every 3 days half amounts change liquid, and coinduction 2 weeks, oil red dyeing identification fat drips were formed.
, through screening the DMEM-HG of FBS, 1 μM of dexamethasone, 200 μM of indocin, IBMX 0.5mM, pancreas is being added containing 10%
Island 10 μ g/ml of element are cultivated 3 days, and morphologic change occurs for cell, are gradually tapered up and are shortened by fusiform fibroblast sample, and 90%
The above cell becomes cube or polygonal；Continuous culture 7 days, has small fat drips to occur, with culture under mirror in visible cell
The extension of time, fat drips are gradually increased and merge, until when cultivating 2 weeks, it is seen that merge pockets of fat drips full of entire cell.Oil red
The fat generated in O dyeing visible cell dyes red by specificity.
(3) at chondrocyte induction
By being separately cultured for embodiment 1, the 3 generations above cell, according to every pipe 2 × 105Cell is dispensed into 15ml polypropylene
Centrifuge tube, low-speed centrifugal make cell form micelle in test tube, and insulin is added in the DMEM-HG containing 2.5%FBS, turns iron
Albumen, sodium selenite each 1.25 μ g/ml of 6.25 μ g/ml, BSA, Sodium Pyruvate 1mM/L, 37.5 μ g/ml of ascorbic acid phosphoric acid,
TGF-β150ng/ml is put in 37 DEG C, 5%CO2, cultivate under saturated humidity, every 3 days half amounts change liquid, continuous culture 2 weeks.
Cell micelle is broken up into smear after inducing 2 weeks, alcian blue (Alcian blue) dyes visible II Collagen Type VI and formed carefully
Extracellular matrix is blue, and control group is contaminated without indigo plant.
6, RT-PCR detects placenta MSC multi-lineage potential
Cell after collecting induction, extracts cell total rna using Trizol reagent, carries out RT-PCR using it as template, instead
Transcription and PCR operation are carried out according to RT-PCR kit specification, Primer, primer sequence, PCR product size, special
Property etc. is as shown in the table 1 of its [0086]~[0087] CN102676451A.The results show that cell can express series after external evoked
Specific mrna: cell expresses PPAR- γ after Adipogenic induction, and cell expresses osteopontin after osteogenic induction
(Osteopontin), at cell expression collagen I I (Collagen II) after chondrocyte induction, illustrate that obtained MSC cell has
Skeletonization, at fat, at cartilage differentiation ability, meet generally acknowledged MSC standard.
By the detection of above series of data target, show the MSC isolated using the method for the present invention, have to
Osteoblast, fat cell, the ability of Chondrocyte Differentiation, it was demonstrated that the MSC that the method for the present invention obtains has stem cell properties.
Embodiment 2 and 3 gained placenta mesenchyma stem cell of embodiment also according to this test example method measurement/processing, as a result with
1 cell results of embodiment are essentially identical.
The foundation of test example 2, placenta stem-cell library
1, the detection of cell activity
The number for freezing front and back living cells is counted using trypan blue staining.
2, the detection of cell contamination
Using a small amount of cell culture, detect cell whether the pollution by fungi and bacterium.Utilize aetology method, detection
Cell whether by Hepatitis B virus, hepatitis, AIDS, cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb,
HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, TRUST infection.
3, the detection of hereditary disease
Using the method for molecular genetics, detecting freeze-stored cell whether there is hereditary disease.
4, HLA-ABC/DR distribution type
Cell HLA-ABC/DR phenotype is detected, and is placed on record.The HLA testing result such as following table of 1 gained cell of embodiment,
Show that MSC is consistent with cord blood cells.
| HLA-A* | HLA-B* | HLA-DRB1* |
| 26:01,33:03 | 15:01,58:01 | 03:01,04:03 |
5, the investigation of cell origin
Fetus and its particulars of parent are recorded, and are placed on record.
6, the foundation of placenta stem-cell database
After saving normal placenta stem-cell, the database of placenta stem-cell is established, including the first six data, and
Foundation is associated with freeze-stored cell.
Embodiment 2 and 3 gained placenta mesenchyma stem cell of embodiment also according to this test example method measurement/processing, as a result with
1 cell results of embodiment are essentially identical.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.
Claims (10)
1. the method for separating mesenchymal stem cell from the blood vessel of placenta, method includes the following steps:
(1) placenta is soaked in 75% alcohol disinfecting 30 seconds, is then rinsed twice with PBS；
(2) placenta blood vessel is separated from the navel baes position of placenta, squeezes blood vessel with surgical forceps to remove blood stains；
(3) scissors for vessels is broken into 1~2mm^3 fragment, is cleaned with PBS, then filter removal residual blood stains with 300 mesh filter screens, obtained
Placenta vascular tissue；
(4) plus mixed enzyme solution digests；
(5) addition serum terminates digestion, then is filtered with 300 mesh filter screens, collects tissue fluid, and PBS washing merges cleaning solution；
(6) centrifugation obtains cell precipitation, is cleaned one time with PBS, is centrifuged, obtains original placenta mesenchyma stem cell (P0 generation),
It is resuspended with DMEM-F12 basal medium, samples and count the quantity and motility rate of karyocyte；Gained cell is with freezing protection liquid
It freezes to recover culture before using again or gained cell then carries out the operation of below step；
(7) make cell inoculation to T75 culture bottle, add complete medium culture；
(8) it changes the liquid once within every 3 days in incubation, until cell confluency is up to 80% or more, passage obtains P1 and fills between placenta
Matter stem cell；
And optional following one or more steps:
(9) cellular identification and/or detection are carried out for placenta mesenchyma stem cell obtained by step (8)；
(10) placenta mesenchyma stem cell after passage obtained by step (8) is frozen in liquid nitrogen；
(11) database of the placenta stem-cell comprising information above is established, and makes the freeze-stored cell of the database Yu step (10)
It is associated.
2. the method according to claim 1, wherein mixed enzyme solution described in step (4) is the PBS buffer solution for being added to mixed enzyme.
3. the method according to claim 1 wherein includes: 0.1~0.3% such as 0.2% in mixed enzyme solution described in step (4)
Such as such as 0.1% deoxyribonuclease I of 0.15% clostridiopetidase A IV, 0.05~0.15% of clostridiopetidase A II, 0.1~0.2%.
4. the method according to claim 1 wherein adds mixed enzyme solution to digest 0.5~2h, such as digestion 1h in step (4).
5. the method according to claim 1, the centrifugation described in step (6) is, for example, with 1000~2000rpm such as 1500rpm
It is centrifuged 3~7min such as 5min.
6. the method according to claim 1, inoculum density is 0.5~2x10^5/cm^2, such as inoculum density in step (7)
For 1x10^5/cm^2.
7. the method according to claim 1, in step (7), the complete medium is consisting of: DMEM-F12+15%FBS
+ 10ng/ml basic fibroblast growth factor.
8. the method according to claim 1, in step (7), the culture is in 37 DEG C, 5%CO2It is cultivated in incubator.
It is according to claim 1~8 described in any one 9. a kind of placenta mesenchyma stem cell isolated from placenta blood vessel
What method obtained.
10. digestive enzyme compositions used in a kind of method from placenta blood vessel isolation of human placenta mesenchymal stem, the digestion
Enzymatic compositions are the PBS buffer solution containing tissue digestion enzyme, and being somebody's turn to do the PBS buffer solution containing tissue digestion enzyme is in PBS buffer solution
Middle addition is selected from following one or more digestive ferments: dispase, pancreatin, deoxyribonuclease I (DNase I), clostridiopetidase A
II, clostridiopetidase A IV, hyaluronidase；For example, including following digestive ferment: deoxyribonuclease I in the digestive enzyme compositions
(DNase I), clostridiopetidase A II, clostridiopetidase A IV；Such as include in the PBS buffer solution containing the digestive ferment: 0.1~0.3%
Such as 0.2% clostridiopetidase A II, 0.1~0.2% such as 0.15% clostridiopetidase A IV, 0.05~0.15% such as 0.1% deoxyribose core
Sour enzyme I；For example, including in the PBS buffer solution containing the digestive ferment: 0.2% clostridiopetidase A II, 0.15% clostridiopetidase A IV, 0.1%
Deoxyribonuclease I.
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