CN107299082A - Placenta interstitial cell and the method for being trained mescenchymal stem cell are separated from tissue - Google Patents
Placenta interstitial cell and the method for being trained mescenchymal stem cell are separated from tissue Download PDFInfo
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- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Abstract
The present invention relates to placenta interstitial cell and the method for being trained mescenchymal stem cell is separated from tissue, mixed enzyme digestive juice used in this method is further related to.Specifically, the described method comprises the following steps：The processing of placental lobules, mixing enzymic digestion and termination, collect primary cell, primary cell freeze, the step such as cell recovery and passage.The efficiency from placenta separating mesenchymal stem cell can be effectively improved using the inventive method.Such as gained primary cell is the purer interstitial cell of a group (CD73 expression is not expressed more than 60%, CD45), and per gram of tissue obtains cell number up to 2.5 × 107, and yield is stable, the P1 P5 through mescenchymal stem cell obtained by Secondary Culture are mescenchymal stem cell for cell, and positive expression CD73, CD90, CD105 are more than 98%, and radiolucent table is less than 2% up to CD34, CD45, CD19, CD11b, HLA DR；P5 is less than 1% for g2 phase cell, and multiplication capacity is strong, is introduced into division stage；Under the stimulation of specific inducing culture, oriented Gegenbaur's cell, lipoblast, the ability of chondroblast differentiation.
Description
Technical field
The present invention relates to the method that stem cell is separated from placenta, the separating mesenchymal stem cell more particularly to from placenta
Method, particularly relates to a kind of digestive enzyme compositions using unique formula of the present invention from placenta tissue to divide
Sow discord cell plastid and the method for being trained mescenchymal stem cell.Can effectively it be improved from placenta compartment using the inventive method
The efficiency of mesenchymal stem cells.
Background technology
The mescenchymal stem cell of mescenchymal stem cell (mesenchymal stem cell, MSC) such as mankind be earliest from
Separated in marrow, have the tissue of multi-lineage potential and self-renewal capacity dry thin from a mesoblastic class
Born of the same parents, in vivo with external specified conditions have to Gegenbaur's cell, 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
New research shows that mescenchymal stem cell has immunological regulation and Hematopoiesis Support affect, and is easy to foreign gene importing expression.
Therefore mescenchymal stem cell not still tissue-engineered bone, cartilage and cardiac muscle build in seed cell, it is important in gene therapy
Carrier cell, and because 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 donor takes marrow to need to undergo the painful operation of a comparison, and had very during materials and after materials
High infection chance；Because the content of MSC in human bone marrow is extremely rare, every 105~106Only about 1 in individual mononuclearcell
It is individual, and with the increase at age, quantity, propagation and the differentiation capability of mescenchymal stem cell are remarkably decreased in marrow, make it
It is restricted in research and application especially clinical practice.Placenta originating from embryonic development period extraembryonic mesoderm be by
Matter, blood vessel and trophocyte composition, contain substantial amounts of mesenchyma composition.Newest research shows dry thin containing what is enriched in placenta
Born of the same parents, be separately cultured out from placenta these multipotential stem cells by for experimental study and clinical practice open up one it is brand-new and abundant
Source.
The existing separation stem cell from placenta still has shortcomings so as to set up the method in placenta stem-cell storehouse, such as pure
Degree is not enough, and/or quantity is not high, and then shows that these methods can not still meet the expectation of people.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 kind of 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 that primary mesenchymal stem cells and serum-free amplification are extracted from placenta ".In addition, Chinese Patent Application No.
201210044648X discloses a kind of method of the separating mesenchymal stem cell from placenta.Purity of these methods in extract
And/or remained to be further improved in terms of the rate of recovery.
This area remains a need for the new method that stem cell is separated from placenta, is particularly efficiently separated from placenta
The method of mescenchymal stem cell.In addition, this area still need new be used for the separating mesenchymal stem cell methods from placenta
The middle digestive enzyme compositions used, to improve the method efficiency of the separating mesenchymal stem cell from placenta.
The content of the invention
Present invention aim to address the not enough practical, simple there is provided one kind of existing acquisition placenta mesenchyma stem cell method
Singly, efficiently separate interstitial cell from placenta tissue and be trained the method for mescenchymal stem cell and optionally set up placenta and do
The method of cell bank.Meanwhile, another object of the present invention is to interstitial cell and be trained for above-mentioned separated from placenta tissue
The method of mescenchymal stem cell provides a kind of digestive enzyme compositions.The inventors discovered that using special operating method and especially
The digestive enzyme compositions of prescription, the cell purity obtained is high and/or cell recoveries are high.The present invention is able to based on this discovery
Complete.
Therefore, first aspect present invention is there is provided the separation interstitial cell from placenta tissue and is trained mescenchymal stem cell
Method, this method comprises the following steps：
(1) processing of placental lobules：Placenta is placed in ceramic whiteware disk, rinsed with tissue-wash solution to remove placenta hemostasis,
Clip 20g placental lobules is organized in steel bowl, is cleaned twice using tissue-wash solution, and is soaked after 5min, weighs 15g preferable
It is organized in 100mm glass dishes；Plus 10ml tissue-wash solutions, leaflet is shredded to 0.2cm3Left and right size, plus 100ml tissue wash
300 mesh filter screens filtering after liquid is stirred evenly, then this operates to be cleaned with tissue-wash solution twice to remove haemocyte repeatedly；
[wherein, described tissue-wash solution is to include 1% 0.9% dual anti-physiological saline]
(2) mixing enzymic digestion and termination：By after cleaning leaflet tissue add 37 DEG C preheating 15~30ml (such as 20~
25ml, such as 23ml) fully mix in mixed enzyme digestive juice after, 37 DEG C of shaking table, 100rpm vibration digestion 30min, digestion terminate
Afterwards, 2ml FBS is added into tissue fluid to terminate digestion；
[wherein, included in described mixed enzyme digestive juice：Hank ' the s balanced salt solutions of 15~30 volumes, 0.2~0.6
The Liberase MNP-S enzymes of volume, (Hank ' the s balance salt of such as 20~25 volumes is molten for the DNA I types enzyme of 0.2~2 volume
Liquid, the Liberase MNP-S enzymes of 0.3~0.5 volume, the DNA I type enzymes of 0.5~1 volume, the Hank ' s of such as 22 volumes are put down
Weigh salting liquid, the Liberase MNP-S enzymes of 0.4 volume, the DNA I types enzyme of 0.7 volume)；The Liberase MNP-S enzymes example
The Liberase MNP-S enzymes of Roche Holding Ag, such as biological purchased from western treasured in this way, its article No.：5578582001]
(3) primary cell is collected：50ml tissue-wash solutions are added in rapid gained tissue fluid one step up, are mixed, 300 mesh mistakes
Filter, collects cell liquid；So wash postdigestive tissue twice repeatedly, filtrate twice is incorporated into centrifuge tube, 1500rpm from
Heart 8min (acceleration 9, deceleration 7)；Remove supernatant, plus appropriate tissue-wash solution is resuspended and is supplemented to 200ml, 1500rpm from
Heart 8min (acceleration 9, deceleration 7)；Supernatant is removed, cell precipitation adds DMEM-F12 to be resuspended to 30ml, 100um strainer filterings
Afterwards, then with 10ml DMEM-F12 flushing filtering nets, 40ml cell suspensions is obtained, primary cell is used as；
[cell suspension of this primary cell can carry out cell count with sysmex blood analysers]
(4) primary cell freezes：Make cell suspension 1800rpm, centrifugation 10min (acceleration 9, deceleration 7), leave and take cell
Precipitation and lower liquid 5ml, are slowly added into frozen stock solution 10ml, side edged shakes up after resuspension；Gained cell suspension is dispensed to 9 2ml
In cryopreservation tube, often pipe 1.5ml, puts in the program temperature reduction box of precooling, is cooled using programmed cooling instrument device program, then cell is transferred to
Frozen in liquid nitrogen storage tank；
[wherein, the formula of the frozen stock solution is：65% DMEM-F12,15% human serum albumins (HSA), 20%
DMSO such as WAK brands DMSO]
(5) cell recovery：Cell is moved to 15ml centrifuge tubes by the cell for taking 2 pipes to freeze, 37 DEG C of quick-thawings, plus 8ml complete
Full culture medium drop recovery；Then 1200rpm is centrifuged after 5min (acceleration 9, deceleration 7), goes supernatant, plus 5ml to cultivate completely
Base is resuspended；It is inoculated in per solencyte in 1 T75 blake bottle, supplement complete medium puts CO2 incubators (37 DEG C, 5% to 30ml
CO2, saturated humidity) middle culture；Liquid was once changed entirely with complete medium every 3-4 days, according to clone's shape after recovering 12 days
Counted into situation, be not less than 3000 cell/cm to cell density2When can carry out ensuing passage；
[wherein, the complete medium is the DMEM-F12 culture mediums for including 10%FBS]
(6) passage：P0 is taken for cell PBS, plus 2ml pancreatin digestion 2-5min, until cell is largely de-
Fall, plus 5ml complete mediums terminate digestion, cell is transferred in centrifuge tube, (acceleration 9 slows down 1400rpm centrifugations 5min
7) degree, abandons counting after supernatant, plus the resuspension of 5ml complete mediums and is seeded to blake bottle, cell density is 8000~12000 thin
Born of the same parents/cm2, put culture in CO2 incubators (37 DEG C, 5%CO2, saturated humidity) and (be generally incubated 5 to cell density up to more than 90%
It or so), complete the passage from P0 generation to P1 generations；Aforesaid operations are repeated in carry out P1 generations respectively to P2 generations, P2 generations
To P3 generations, P3 generations to the passage in P4 generations, P4 generation to P5 generations, each generation mescenchymal stem cell is obtained.
The method of any embodiment according to a first aspect of the present invention, wherein also including：
At least one of (7) placenta mesenchyma stem cell obtained by step (6), detection following items：It is cytoactive, thin
Born of the same parents' pollution, hereditary disease, HLA-ABC/DR distribution type.
The method of any embodiment according to a first aspect of the present invention, wherein also including：
(8) each after passing on obtained by step (6) freezes for placenta mesenchyma stem cell in liquid nitrogen.
The method of any embodiment according to a first aspect of the present invention, wherein also including：
(9) database of the placenta stem-cell comprising information above is set up, and makes the database and freezing for step (8) thin
Born of the same parents are associated.
The method of any embodiment according to a first aspect of the present invention, wherein gained is each for the thin of placenta mesenchyma stem cell
Born of the same parents' purity is more than 90%.In one embodiment, after the placenta mesenchyma stem cell more than generation is passed on through 3, cell purity
More than 95%.
The method of any embodiment according to a first aspect of the present invention, wherein Hank's balanced salt solutions composition is：
8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO47H2O, 0.1g/L MgCl26H2O, 0.06g/L's
Na2HPO42H2O, 0.06g/L KH2PO4,1.0g/L glucose, 0.14g/L CaCl2,0.35g/L NaHCO3,
The phenol red of 0.2g/L, hydrochloric acid or sodium hydroxide regulation pH to 7.4.The method of any embodiment according to a first aspect of the present invention,
In wherein described mixed enzyme digestive juice in addition to comprising Hank's balanced salt solutions, Liberase MNP-S enzymes, DNA I type enzymes,
Also it is added with 0.2~0.3g/L zinc chloride.It has been had now surprisingly been found that, using the mixing for adding this concentration range zinc chloride
In the case of enzymic digestion liquid, the CD73 expression of gained primary cell is not expressed more than 60%, CD45, and in gained primary cell
Mescenchymal stem cell content reach 60%-70%, show high concentration of stem cells；And work as in mixed enzyme digestive juice and be not added with this
During zinc chloride, the mescenchymal stem cell content in primary cell is less than 38%, generally in the range of 31~38%.
The method of any embodiment according to a first aspect of the present invention, wherein the cytoactive detection is to utilize trypan blue
Decoration method counts the number for freezing front and rear living cells.
The method of any embodiment according to a first aspect of the present invention, wherein cell contamination detection utilizes a small amount of cell
Whether culture, detection cell is polluted by fungi and bacterium.In one embodiment, the cell contamination detection is to utilize
Aetology method, whether detection cell is by selected from one or more following of infection：Hepatitis B virus, hepatitis, AIDS
Poison, cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-
IgM and EBV-IgA, TRUST.
The method of any embodiment according to a first aspect of the present invention, wherein hereditary disease detection is to utilize molecular genetic
Method, detection freeze-stored cell whether there is hereditary disease.
The method of any embodiment according to a first aspect of the present invention, wherein the HLA-ABC/DR distribution type is detection cell
HLA-ABC/DR phenotypes.
The method of any embodiment according to a first aspect of the present invention, wherein the placenta mesenchyma stem cell is through program
Temperature-fall period is frozen in liquid nitrogen.
The method of any embodiment according to a first aspect of the present invention, wherein the database include with preserved it is thin
All related data of born of the same parents, include but is not limited to：The biological characteristics testing result of cell, multi-lineage potential qualification result,
Cellular elements genetic diagnosis result, fetus and its particulars of father and mother.
In addition, there is provided a kind of placenta mesenchyma stem cell in first aspect present invention method.Therefore the present invention second
Aspect provides a kind of placenta mesenchyma stem cell.
Placenta mesenchyma stem cell according to a second aspect of the present invention, it is any embodiment party according to a first aspect of the present invention
What case methods described was obtained.
Placenta mesenchyma stem cell according to a second aspect of the present invention, the cell of the placenta mesenchyma stem cell in its each generation is pure
Degree is more than 90%.In one embodiment, after the placenta mesenchyma stem cell more than generation is passed on through 3, cell purity is more than
95%.
Further, third aspect present invention provides one kind and filled between interstitial cell is separated from placenta tissue and is trained
In the mixed enzyme digestive juice used in the method for matter stem cell, the mixed enzyme digestive juice comprising Hank's balanced salt solutions,
Liberase MNP-S enzymes, DNA I type enzymes.
The mixed enzyme digestive juice of any embodiment according to a third aspect of the present invention, including：15~30 volumes
Hank ' s balanced salt solutions, the Liberase MNP-S enzymes of 0.2~0.6 volume, the DNA I types enzyme (such as 20 of 0.2~2 volume
Hank ' the s balanced salt solutions of~25 volumes, the Liberase MNP-S enzymes of 0.3~0.5 volume, the DNA I types of 0.5~1 volume
Hank ' the s balanced salt solutions of enzyme, such as 22 volumes, the Liberase MNP-S enzymes of 0.4 volume, the DNA I types of 0.7 volume
Enzyme).
The mixed enzyme digestive juice of any embodiment according to a third aspect of the present invention, wherein the Hank's balanced salt solutions
Constitute and be：8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO47H2O, 0.1g/L MgCl26H2O,
0.06g/L Na2HPO42H2O, 0.06g/L KH2PO4,1.0g/L glucose, 0.14g/L CaCl2,0.35g/L
The phenol red of NaHCO3,0.2g/L, hydrochloric acid or sodium hydroxide regulation pH to 7.4.Any embodiment party according to a third aspect of the present invention
The mixed enzyme digestive juice of case, wherein in addition to comprising Hank's balanced salt solutions, Liberase MNP-S enzymes, DNA I type enzymes, also
Zinc chloride added with ormal weight as described herein.It has been had now surprisingly been found that, using this concentration range zinc chloride of addition
Mixed enzyme digestive juice in the case of excellent technique effect as described herein can be presented.
The mixed enzyme digestive juice of any embodiment according to a third aspect of the present invention, wherein described separate from placenta tissue
Interstitial cell and the method for being trained mescenchymal stem cell, it comprises the following steps：
(1) processing of placental lobules：Placenta is placed in ceramic whiteware disk, rinsed with tissue-wash solution to remove placenta hemostasis,
Clip 20g placental lobules is organized in steel bowl, is cleaned twice using tissue-wash solution, and is soaked after 5min, weighs 15g preferable
It is organized in 100mm glass dishes；Plus 10ml tissue-wash solutions, leaflet is shredded to 0.2cm3Left and right size, plus 100ml tissue wash
300 mesh filter screens filtering after liquid is stirred evenly, then this operates to be cleaned with tissue-wash solution twice to remove haemocyte repeatedly；
[wherein, described tissue-wash solution is to include 1% 0.9% dual anti-physiological saline]
(2) mixing enzymic digestion and termination：By after cleaning leaflet tissue add 37 DEG C preheating 15~30ml (such as 20~
25ml, such as 23ml) fully mix in mixed enzyme digestive juice after, 37 DEG C of shaking table, 100rpm vibration digestion 30min, digestion terminate
Afterwards, 2ml FBS is added into tissue fluid to terminate digestion；
[wherein, included in described mixed enzyme digestive juice：Hank ' the s balanced salt solutions of 15~30 volumes, 0.2~0.6
The Liberase MNP-S enzymes of volume, (Hank ' the s balance salt of such as 20~25 volumes is molten for the DNA I types enzyme of 0.2~2 volume
Liquid, the Liberase MNP-S enzymes of 0.3~0.5 volume, the DNA I type enzymes of 0.5~1 volume, the Hank ' s of such as 22 volumes are put down
Weigh salting liquid, the Liberase MNP-S enzymes of 0.4 volume, the DNA I types enzyme of 0.7 volume)；The Liberase MNP-S enzymes example
The Liberase MNP-S enzymes of Roche Holding Ag, such as biological purchased from western treasured in this way, its article No.：5578582001]
(3) primary cell is collected：50ml tissue-wash solutions are added in rapid gained tissue fluid one step up, are mixed, 300 mesh mistakes
Filter, collects cell liquid；So wash postdigestive tissue twice repeatedly, filtrate twice is incorporated into centrifuge tube, 1500rpm from
Heart 8min (acceleration 9, deceleration 7)；Remove supernatant, plus appropriate tissue-wash solution is resuspended and is supplemented to 200ml, 1500rpm from
Heart 8min (acceleration 9, deceleration 7)；Supernatant is removed, cell precipitation adds DMEM-F12 to be resuspended to 30ml, 100um strainer filterings
Afterwards, then with 10ml DMEM-F12 flushing filtering nets, 40ml cell suspensions is obtained, primary cell is used as；
[cell suspension of this primary cell can carry out cell count with sysmex blood analysers]
(4) primary cell freezes：Make cell suspension 1800rpm, centrifugation 10min (acceleration 9, deceleration 7), leave and take cell
Precipitation and lower liquid 5ml, are slowly added into frozen stock solution 10ml, side edged shakes up after resuspension；Gained cell suspension is dispensed to 9 2ml
In cryopreservation tube, often pipe 1.5ml, puts in the program temperature reduction box of precooling, is cooled using programmed cooling instrument device program, then cell is transferred to
Frozen in liquid nitrogen storage tank；
[wherein, the formula of the frozen stock solution is：65% DMEM-F12,15% human serum albumins (HSA), 20%
DMSO such as WAK brands DMSO]
(5) cell recovery：Cell is moved to 15ml centrifuge tubes by the cell for taking 2 pipes to freeze, 37 DEG C of quick-thawings, plus 8ml complete
Full culture medium drop recovery；Then 1200rpm is centrifuged after 5min (acceleration 9, deceleration 7), goes supernatant, plus 5ml to cultivate completely
Base is resuspended；It is inoculated in per solencyte in 1 T75 blake bottle, supplement complete medium puts CO2 incubators (37 DEG C, 5% to 30ml
CO2, saturated humidity) middle culture；Liquid was once changed entirely with complete medium every 3-4 days, according to clone's shape after recovering 12 days
Counted into situation, be not less than 3000 cell/cm to cell density2When can carry out ensuing passage；
[wherein, the complete medium is the DMEM-F12 culture mediums for including 10%FBS]
(6) passage：P0 is taken for cell PBS, plus 2ml pancreatin digestion 2-5min, until cell is largely de-
Fall, plus 5ml complete mediums terminate digestion, cell is transferred in centrifuge tube, (acceleration 9 slows down 1400rpm centrifugations 5min
7) degree, abandons counting after supernatant, plus the resuspension of 5ml complete mediums and is seeded to blake bottle, cell density is 8000~12000 thin
Born of the same parents/cm2, put culture in CO2 incubators (37 DEG C, 5%CO2, saturated humidity) and (be generally incubated 5 to cell density up to more than 90%
It or so), complete the passage from P0 generation to P1 generations；Aforesaid operations are repeated in carry out P1 generations respectively to P2 generations, P2 generations
To P3 generations, P3 generations to the passage in P4 generations, P4 generation to P5 generations, each generation mescenchymal stem cell is obtained.
The mixed enzyme digestive juice of any embodiment according to a third aspect of the present invention, wherein described separate from placenta tissue
Interstitial cell and being trained in the method for mescenchymal stem cell also includes：
At least one of (7) placenta mesenchyma stem cell obtained by step (6), detection following items：It is cytoactive, thin
Born of the same parents' pollution, hereditary disease, HLA-ABC/DR distribution type.
The mixed enzyme digestive juice of any embodiment according to a third aspect of the present invention, wherein described separate from placenta tissue
Interstitial cell and being trained in the method for mescenchymal stem cell also includes：
(8) each after passing on obtained by step (6) freezes for placenta mesenchyma stem cell in liquid nitrogen.
The mixed enzyme digestive juice of any embodiment according to a third aspect of the present invention, wherein described separate from placenta tissue
Interstitial cell and being trained in the method for mescenchymal stem cell also includes：
(9) database of the placenta stem-cell comprising information above is set up, and makes the database and freezing for step (8) thin
Born of the same parents are associated.
The mixed enzyme digestive juice of any embodiment according to a third aspect of the present invention, wherein described separate from placenta tissue
Interstitial cell and be trained cytoactive detection described in the method for mescenchymal stem cell be using trypan blue staining count freeze
Deposit the number of front and rear living cells.
The mixed enzyme digestive juice of any embodiment according to a third aspect of the present invention, wherein described separate from placenta tissue
Interstitial cell is simultaneously trained the detection of cell contamination described in the method for mescenchymal stem cell using a small amount of cell culture, detects cell
Whether polluted by fungi and bacterium.In one embodiment, the cell contamination detection is to utilize aetology method, inspection
Cell is surveyed whether by selected from one or more following of 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。
The mixed enzyme digestive juice of any embodiment according to a third aspect of the present invention, wherein described separate from placenta tissue
Interstitial cell and to be trained the detection of hereditary disease described in the method for mescenchymal stem cell be the method using molecular genetics, detection
Freeze-stored cell whether there is hereditary disease.
The mixed enzyme digestive juice of any embodiment according to a third aspect of the present invention, wherein described separate from placenta tissue
Interstitial cell and be trained HLA-ABC/DR distribution type described in the method for mescenchymal stem cell be detection cell HLA-ABC/DR tables
Type.
The mixed enzyme digestive juice of any embodiment according to a third aspect of the present invention, wherein described separate from placenta tissue
Interstitial cell and to be trained placenta mesenchyma stem cell described in the method for mescenchymal stem cell frozen through program temperature-fall period
In liquid nitrogen.
The mixed enzyme digestive juice of any embodiment according to a third aspect of the present invention, wherein described separate from placenta tissue
Interstitial cell and be trained database described in the method for mescenchymal stem cell include it is all related to the cell preserved
Data, include but is not limited to：The biological characteristics testing result of cell, multi-lineage potential qualification result, cellular elements heredity
Learn diagnostic result, fetus and its particulars of father and mother.
In the above-mentioned various operating procedures of the present invention, although its describe specific steps are in some details or language is retouched
The step of stating described in the preparation example with following detailed description part is otherwise varied, however, those skilled in the art
Approach described above step can be summarized according to the detailed disclosure of full text of the present invention completely.
Any embodiment of the either side of the present invention, can be combined with other embodiments, as long as they are not
Contradiction occurs.In addition, in any embodiment of either side of the present invention, any technical characteristic goes for other realities
The technical characteristic in scheme is applied, as long as they are not in contradiction.The invention will be further described below.
All documents recited in the present invention, their full content is incorporated herein by reference, and if these are literary
Offer expressed implication with it is of the invention inconsistent when, be defined by the statement of the present invention.In addition, the various terms that use of the present invention and
Phrase has well known to a person skilled in the art general sense, nonetheless, the present invention remain desirable at this to these terms and
Phrase is described in more detail and explained that the term and phrase referred to is if any inconsistent with common art-recognized meanings, with institute's table of the present invention
The implication stated is defined.
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 linguistic context of the invention, term " placenta mesenchyma stem cell " can with " placenta stem-cell ",
" stem cell ", " mescenchymal stem cell " used interchangeably, unless otherwise clearly indicating.
In the present invention, term " PBS " or " PBS " refer to phosphate buffer.Those skilled in the art are ripe
Know the PBS used under situation of the present invention general formula and compound method and their general aspects such as pH value or pH
Scope, and these PBSs generally can be by the pre-mixing liquor (or prewired powder) of commercial sources acquisition, such as this
The PBS of invention field is typically the PBS of pH7.4 (± 0.1) commercialization buffer solution, such as HyClone brands；Ability
PBS composition during the application of domain classics includes 137mM sodium chloride, 2.7nM potassium chloride and 10mM phosphate radicals, in this hair
In bright if not otherwise specified, composition when PBS used is used is the composition.
In the present invention, term " placenta " refers to newborn fetal placenta, particularly relates to the placenta within 4 hours postpartum.
The present inventor was once separately cultured mescenchymal stem cell using perfusion method from placenta, obtained purity very high
Mescenchymal stem cell.But a large amount of stem cells are still suffered from after perfusion and are trapped in placenta tissue, it is impossible to effectively by
Separate.It is understood that mescenchymal stem cell can not be obtained to greatest extent using perfusion method.
The invention discloses a kind of method of separating mesenchymal stem cells a large amount of from placenta, and profit is preserved in this way
Placenta mesenchyma stem cell simultaneously sets up placenta stem-cell storehouse.It is dry thin that the present inventor is separately cultured mesenchyma in the past in summary
It is successful from placenta with reference to stationary culture using Various Tissues digestive ferment mixture slaking placental lobules tissue block on the basis of born of the same parents
In isolated a large amount of mescenchymal stem cells.Mescenchymal stem cell purity that the inventive method is obtained is high, quantity is more, has and bone
Bone marrow-drived mesenchymal stem identical biological characteristics, can be thin to Gegenbaur's cell, cartilage cell, fat cell, endothelial cell, nerve
Born of the same parents etc. break up.Because stem cell is inmature compared with adult stem cell in placenta, rich content, before clinically having a wide range of applications
Scape, we freeze mescenchymal stem cell with conventional cell freezing method as bleeding of the umbilicus, set up placenta stem-cell
Storehouse, is that the further investigation of later stem cell and clinical treatment lay the foundation.
Because, containing abundant candidate stem cell, people set up unbilical blood bank, and umbilical hemopoietic stem cell, this is important in bleeding of the umbilicus
Living resources store, be that a variety of diseases in the blood system and disease of immune system provide a kind for the treatment of means.Same placenta
Mescenchymal stem cell is as a kind of more importantly stem cell resource, and we are freezed with conventional cell freezing method
Preserved for a long time in -196 degrees Celsius of profound hypothermia liquid nitrogen, set up placenta stem-cell storehouse, be stem-cell therapy preservation kind in the future
Son.
Especially, it should be noted that, by the inventive method, mescenchymal stem cell very high purity can be obtained in P0 generations
Primary cell, the CD73 expression of these primary cells is not expressed more than 60%, CD45, and mesenchyma is done in these primary cells
Cell content reaches 60%-70%.
Having the technical effect that for the method for the present invention is obvious.For example, the present invention chooses mature placental samples, clip
Placental lobules ad-hoc location tissue 15g, it is digested, obtain being purified after cell, you can obtain with one kind mixing enzyme system
The purer interstitial cell of a group (CD73 expression do not expressed more than 60%, CD45), per gram of tissue acquisition cell number up to 2.5 ×
107, and yield is stable, greatly reduces sample specificity.Recover and cultivate after this batch of primary interstitial cell is frozen, use classics
Complete medium formula cultivated, 4 days or so can microscopy to more spindle-type attached cell, cell confluency reaches within 10 days
To 70-80%, you can pass to P1 generations.After continuous passage to P5 generations, streaming phenotypic evaluation, cell cycle detection and induction point are carried out
The experiment such as change, it is mescenchymal stem cell for cell as a result to show P1-P5, and positive expression (CD73, CD90, CD105) is more than
98%, radiolucent table is less than 2% up to (CD34, CD45, CD19, CD11b, HLA-DR)；P5 is less than 1% for g2 phase cell, increases
Grow ability strong, be introduced into division stage；Under the stimulation of specific inducing culture, oriented Gegenbaur's cell, lipoblast is thin into cartilage
The ability of born of the same parents' differentiation.
The present invention is simple to operate, convenient and practical, can obtain substantial amounts of mescenchymal stem cell, and differentiation performance is good, with into
The ability of the cell differentiations such as osteocyte, fat cell, cartilage cell, endothelial cell, nerve cell.With the comparison of existing method：
Current MSC mainly extracts donor bone marrow or perfusion method separation placenta using modus operandi, and adhere-wall culture is obtained.The method gets cell number
Amount is few, and donor has the possibility of infection after taking marrow neutralization to take marrow.Present invention success separation from placenta obtains a large amount of pure
The higher mescenchymal stem cell of degree, and set up placenta stem-cell storehouse to lay in the dry thin of this great application prospect with this method
Born of the same parents.The method is simple and easy to do, and because placenta is as bleeding of the umbilicus, Cell Component is inmatureer, and wide material sources are conveniently easy to get, therefore this
The method of invention will have extensive prospect in the clinical practice of stem cell.
Brief description of the drawings
Fig. 1：The streaming phenotypic evaluation result figure of gained primary cell of the invention.
Fig. 2：Micrograph of the sample in P0 succeeding generations.
Fig. 3：Micrograph of the sample in P5 succeeding generations.
Fig. 4：Sample is in P5 for streaming phenotypic evaluation result.
Fig. 5：DNA content-cell number graphs of a relation of the sample P5 for cell.
Fig. 6：P5 breaks up for the induction of cell to be tested, and shows that it has the energy broken up to skeletonization, into fat, chondroblast
Power.
Embodiment
The present invention can be further described by the following examples, however, the scope of the present invention is not limited
In following embodiments.One of skill in the art, can be with it is understood that on 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 general to the material and test method that are arrived used in experiment
And/or specific description.Although for realize many materials used in the object of the invention and operating method be it is known in the art that
But the present invention still makees description as detailed as possible herein.
The full cell processing of embodiment 1, placenta：
1st, the preformulation of synthase digestive juice is mixed：Pipette respectively 22ml calcic magnesium ions HBSS (Hank's balance salt it is molten
Liquid), 0.4ml Roche Liberase MNP-S enzymes are (such as purchased from western precious biological, article No.：5578582001), 0.7ml DNA I
Type enzyme is in 50ml centrifuge tubes, then adds zinc chloride (addition concentration is 0.2g/L, 0.25g/L or 0.3g/L), is mixed, 37 DEG C
Preheat more than 20min.The Hank's balanced salt solutions are constituted：8.0g/L NaCl, 0.4g/L KCl, 0.1g/L's
MgSO47H2O, 0.1g/L MgCl26H2O, 0.06g/L Na2HPO42H2O, 0.06g/L KH2PO4,1.0g/L
Glucose, the phenol red of 0.14g/L CaCl2,0.35g/L NaHCO3,0.2g/L, hydrochloric acid or sodium hydroxide adjust pH to
7.4。
2nd, the preparation of placental lobules：Placenta is taken out from collection bag in ceramic whiteware disk, after tissue-wash solution is rinsed, tire is removed
Disk hemostasis, a small amount of placental lobules tissue (20g or so) of clip is in steel bowl.Use tissue-wash solution (0.9% physiological saline+bis-
Anti- (dual anti-is mycillin, content 1%)) clean twice, and soak after 5min, weigh 15g ± 1g and be preferably organized in 100mm
In glass dish.
3rd, the removal of haemocyte：Plus 10ml tissue-wash solutions, leaflet is shredded to 0.2cm3Left and right size, plus 100ml tissues
300 mesh filter screens filtering after cleaning fluid is stirred evenly, then clean with tissue-wash solution and twice (leaflet tissue is moved on in steel bowl added every time
After 100ml tissue-wash solutions are stirred evenly, the filtering of 300 mesh).
4th, mixing enzymic digestion and termination：Leaflet tissue after cleaning is added and filled in warmed-up 23ml mixed enzyme digestive juices
Divide after mixing, 37 degree of 100rpm vibration digestion 30min of shaking table.After digestion terminates, tissue fluid+2ml FBS are terminated.
5th, the collection of primary cell：
Plus the dilution of 50ml tissue-wash solutions mixes tissue fluid, the filtering of 300 mesh is collected cell liquid, washed twice postdigestive
Tissue (every time using 50ml tissue-wash solutions), filtrate is incorporated into 1 250ml centrifuge tube, and 1500rpm centrifugations 8min (accelerates
Degree 9, deceleration 7)；
Supernatant is removed, plus appropriate tissue-wash solution is resuspended and is supplemented to 200ml, (acceleration 9, subtracts 1500rpm centrifugations 8min
Speed 7)；
Supernatant is removed, cell precipitation adds DMEM-F12 to be resuspended to 30ml, after 100um strainer filterings, then with 10ml DMEM-
F12 flushing filtering nets, obtain 40ml cell suspensions, are used as primary cell；1ml suspensions are taken, are carried out for sysmex blood analysers thin
Born of the same parents count, and after measured, the primary cell purity is higher, and mescenchymal stem cell content is about 60%-70%.
6th, primary cell freezes：
Now match somebody with somebody frozen stock solution, the formula of frozen stock solution is：65% DMEM-F12,15% human serum albumins (HSA), 20%
DMSO such as WAK brands DMSO, matching while using；
Make cell suspension 1800rpm, centrifugation 10min (acceleration 9, deceleration 7), leave and take cell precipitation and lower liquid 5ml is (another
Supernatant 10ml keeps sample), the frozen stock solution prepared is slowly added into after resuspension, side edged shakes up；
Cell suspension is dispensed into 9 2ml cryopreservation tubes, often pipe 1.5ml (program temperature reduction box precooling).Remaining cell suspension
+ the supernatant that keeps sample is used for Sterility testing；
Cooled using programmed cooling instrument device program, cell is transferred in liquid nitrogen storage tank, gained primary cell is frozen.
By the full cell processing procedure of the placenta of above-described embodiment 1, mature placental samples are chosen, clip placental lobules is special
Tissue 15g is put in positioning, is digested it with described mixed enzyme digestive juice system, is obtained being purified after cell, you can obtain one
Mesenchyma in the purer primary interstitial cell of group (CD73 expression is not expressed more than 60%, CD45), these primary cells is dry thin
Born of the same parents' content reaches 60%-70%, and the primary cell number that every gram of placental lobules tissue is obtained is up to (2.4~2.8) × 107It is individual, and
Rate is stable, greatly reduces sample specificity.However, when being not added with this zinc chloride in the mixed enzyme digestive juice, it is primary thin
Mescenchymal stem cell content in born of the same parents is less than 38%, generally in the range of 31~38%, and every gram of placental lobules tissue is obtained
Primary cell number be less than 5 × 105It is individual；In addition, the present inventor sends out when carrying out primary cell preparation with reference to other prior arts
The primary cell number that now every gram of placental lobules tissue is obtained is less than 2 × 106It is individual, it is less than the 1/10 of the inventive method.The present invention
It is above-mentioned that primary interstitial cell can be obtained efficiently to the full cell processing of placenta progress, it is to resume to be commissioned to train thereafter to form with pole
The mescenchymal stem cell of high medical value lays a good foundation.
For example, in the present embodiment, cell yield is highly stable after placenta tissue processing, and the typical data of some experiments is shown in
Table 1.
Table 1：The cell yield of primary cell is obtained from placenta tissue
| Handle the date | Sample registered number | Tissue mass (g) | Cell number (× 108) | Cell yield (108/g) |
| 2016.7.22 | 9004116082279 | 15 | 3.8 | 0.25 |
| 2016.7.25 | 9004116082301 | 15.9 | 3.9 | 0.25 |
| 2016.7.26 | 9004116082311 | 14.9 | 3.8 | 0.26 |
| 2016.7.27 | 9004116082319 | 14.9 | 3.8 | 0.26 |
| 2016.8.25 | 9004116082539 | 15 | 3.75 | 0.25 |
In addition, after the processing of above-mentioned placenta gained primary cell streaming phenotypic evaluation result show CD73 expression up to 60% with
On, CD45 is not expressed, and it is the purer interstitial cell of a group to show primary cell, without haemocyte.Streaming phenotypic evaluation result
As shown in Figure 1.
Embodiment 2, primary cell recovery and Secondary Culture
1st, cell recovery：
Cell is moved to 15ml centrifuge tubes, plus 8ml complete medium drops by the cell for taking 2 pipes to freeze, 37 DEG C of quick-thawings
Recovery；If not otherwise indicated, complete medium used herein is the DMEM-F12 culture mediums for including 10%FBS；
Then 1200rpm is centrifuged after 5min (acceleration 9, deceleration 7), goes supernatant, plus 5ml complete mediums to be resuspended；Often
Solencyte is inoculated in 1 T75 blake bottle, and supplement complete medium puts CO2 incubators (37 DEG C, 5%CO2, saturation to 30ml
Humidity) middle culture；Once changed liquid entirely with complete medium every 3-4 days, recovery is entered after 12 days according to Clone formation situation
Row is counted, and is not less than 3000 cell/cm to cell density2When can carry out ensuing passage；
2nd, passage：The P0 after recovery is taken for cell PBS, plus 2ml pancreatin digestion 2-5min, until cell is big
Partial exfoliation, plus 5ml complete mediums terminate digestion, cell is transferred in centrifuge tube, 1400rpm centrifugation 5min (acceleration
9, deceleration 7), abandon counting after supernatant, plus the resuspension of 5ml complete mediums and be seeded to blake bottle, cell density is 8000~12000
Individual cell/cm2, put culture in CO2 incubators (37 DEG C, 5%CO2, saturated humidity) and (generally trained to cell density up to more than 90%
Support 5 days or so), complete the passage from P0 generation to P1 generations；
The passage for being repeated in above-mentioned P0 generation to P1 generations is operated, to carry out P1 generations to P2 generations, P2 generations to P3 generations, P3 generations respectively
To the passage in P4 generations, P4 generation to P5 generations, each generation mescenchymal stem cell is obtained.
In the present embodimentPrimary inoculum density is about 5 × 105cells/cm2, 4 clear water surfaces of inoculation, which are inspected in open country, many patches
Parietal cell, in spindle-type.P1 generations can be reached within 10 days after inoculation.High cell growth speed, amount is more, and form is in fusiformis, full.From
In P0 generations, the cell count exemplary results of some experiments were as shown in table 2 below into the succeeding generations in P1 generations.Wherein PS162279 samples
Originally the micrograph in succeeding generations is as shown in Figure 2.
Table 2：Cell counts of the P0 generations into the succeeding generations in P1 generations
| Sample registered number | P0-P1 | P0 is counted |
| PS162311 | D10 | 8*105(ADAM) |
| PS162319 | D10 | 1.4*106(ADAM) |
| PS162279 | D9 | 1.5*106(ADAM) |
In addition, in P1-P5 inoculation succeeding generations, being generally incubated and harvesting within 4~5 days and be passaged to the next generation.It is exemplary
, micrograph of the PS162279 samples in P5 succeeding generations is as shown in Figure 3.
In this experiment, the streaming phenotypic evaluation in P1-P5 generations is carried out, has as a result been shown, CD73, CD90, CD105 positive table
Up to equal>98%, while identifying CD34, CD45, CD19, HLA-DR, as a result as shown in table 3, these results prove to divide in placenta
It is mescenchymal stem cell from the cell turned out, and purity is high.
Table 3：Streaming phenotypic evaluation results of the P1-P5 for cell
Exemplary, PS162279 samples P5 is as shown in Figure 4 for streaming phenotypic evaluation result.
In addition, for some samples P5 for their growth cycle of raji cell assay Raji, as a result show G2 phase cells<1%, S
Phase cell>10%, it was demonstrated that these ability of cell proliferation are strong, are introduced into division stage, concrete outcome is shown in Table 4.
Table 4：P5 is for cell growth cycle measurement result
| Sample registered number | The GO/G1 phases | The S phases | The G2/M phases |
| PS162311-P5 | 84.60% | 14.80% | 0.64% |
| PS162319-P5 | 82.30% | 16.80% | 0.93% |
| PS162279-P5 | 87.00% | 11.90% | 0.80% |
In addition, for PS162311-P5 cells, drawing its DNA content-cell number graph of a relation, typical result is shown in
Fig. 5
The Identification of Biological Characteristics of embodiment 3, placenta MSC
With reference to authorized patentCN102676451A its [0062] to [0089] method carries out placenta mesenchyma and done
The Identification of Biological Characteristics of cell, as a result shows, using the isolated MSC of the inventive method, with to Gegenbaur's cell, fat
The ability of cell, Chondrocyte Differentiation, it was demonstrated that the MSC that the inventive method is obtained has stem cell properties.
For example, exemplary, induction differentiation test is carried out for cell to P5, as a result show these cells have to skeletonization,
The ability broken up into fat, chondroblast.The typical micrograph into fat differentiation, Osteoblast Differentiation and into cartilage differentiation is shown in Fig. 6.
Embodiment 4, the foundation in placenta stem-cell storehouse
1st, the detection of cytoactive
The number for freezing front and rear living cells is counted using trypan blue staining.
2nd, the detection of cell contamination
Using a small amount of cell culture, whether detection cell is polluted 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.
3rd, the detection of hereditary disease
Using the method for molecular genetics, detection freeze-stored cell whether there is hereditary disease.
4th, HLA-ABC/DR distribution type
Cell HLA-ABC/DR phenotypes are detected, and are placed on record.
5th, the investigation of cell derived
Fetus and its particulars of father and mother are recorded, and are placed on record.
6th, the foundation of placenta stem-cell database
After normal placenta stem-cell is preserved, the database of placenta stem-cell is set up, including the first six data, and
Foundation is associated with freeze-stored cell.
Claims (10)
1. separating interstitial cell and the method for being trained mescenchymal stem cell from placenta tissue, this method comprises the following steps：
(1) processing of placental lobules：Placenta is placed in ceramic whiteware disk, is rinsed to remove placenta hemostasis, clip with tissue-wash solution
20g placental lobules is organized in steel bowl, is cleaned twice using tissue-wash solution, and is soaked after 5min, is weighed 15g and is preferably organized
In 100mm glass dishes；Plus 10ml tissue-wash solutions, leaflet is shredded to 0.2cm3Left and right size, plus 100ml tissue-wash solutions are stirred
300 mesh filter screens filtering after even, then this operates to be cleaned with tissue-wash solution twice to remove haemocyte repeatedly；
(2) mixing enzymic digestion and termination：By after cleaning leaflet tissue add 37 DEG C preheating 15~30ml (such as 20~
25ml, such as 23ml) fully mix in mixed enzyme digestive juice after, 37 DEG C of shaking table, 100rpm vibration digestion 30min, digestion terminate
Afterwards, 2ml FBS is added into tissue fluid to terminate digestion；
(3) primary cell is collected：50ml tissue-wash solutions are added in rapid gained tissue fluid one step up, are mixed, the filtering of 300 mesh,
Collect cell liquid；So wash postdigestive tissue twice repeatedly, filtrate twice is incorporated into centrifuge tube, 1500rpm centrifugations
8min (acceleration 9, deceleration 7)；Supernatant is removed, plus appropriate tissue-wash solution is resuspended and is supplemented to 200ml, 1500rpm centrifugations
8min (acceleration 9, deceleration 7)；Remove supernatant, cell precipitation adds DMEM-F12 to be resuspended to 30ml, after 100um strainer filterings,
10ml DMEM-F12 flushing filtering nets are used again, are obtained 40ml cell suspensions, are used as primary cell；
(4) primary cell freezes：Make cell suspension 1800rpm, centrifugation 10min (acceleration 9, deceleration 7), leave and take cell precipitation
And lower liquid 5ml, frozen stock solution 10ml is slowly added into after resuspension, side edged shakes up；Gained cell suspension is dispensed to 9 2ml and frozen
Guan Zhong, often pipe 1.5ml, puts in the program temperature reduction box of precooling, is cooled using programmed cooling instrument device program, then cell is transferred into liquid nitrogen
Frozen in storage tank；
(5) cell recovery：The cell for taking 2 pipes to freeze, 37 DEG C of quick-thawings, 15ml centrifuge tubes are moved to by cell, plus 8ml is trained completely
Support basic point drop recovery；Then 1200rpm is centrifuged after 5min (acceleration 9, deceleration 7), removes supernatant, plus 5ml complete medium weights
It is outstanding；Be inoculated in 1 T75 blake bottle per solencyte, supplement complete medium to 30ml, put CO2 incubators (37 DEG C, 5%CO2,
Saturated humidity) middle culture；Liquid was once changed entirely with complete medium every 3-4 days, according to Clone formation feelings after recovering 12 days
Condition is counted, and is not less than 3000 cell/cm to cell density2When can carry out ensuing passage；
(6) passage：P0 is taken for cell PBS, plus 2ml pancreatin digestion 2-5min, until cell largely comes off, plus
5ml complete mediums terminate digestion, cell is transferred in centrifuge tube, 1400rpm centrifugation 5min (acceleration 9, deceleration 7),
Abandon to count after supernatant, plus the resuspension of 5ml complete mediums and be seeded to blake bottle, cell density is 8000~12000 cell/cm2,
Culture in CO2 incubators (37 DEG C, 5%CO2, saturated humidity) is put, to cell density up to more than 90%, to complete from P0 generation to P1 generations
Passage；Aforesaid operations are repeated in carry out P1 generations to P2 generations, P2 generations to P3 generations, P3 generations to P4 generations, P4 generations respectively extremely
The passage in P5 generations, obtains each generation mescenchymal stem cell.
2. method according to claim 1, described tissue-wash solution is to include 1% 0.9% dual anti-physiological saline.
3. included in method according to claim 1, described mixed enzyme digestive juice：Hank ' the s balance salt of 15~30 volumes is molten
Liquid, the Liberase MNP-S enzymes of 0.2~0.6 volume, DNA I types enzyme (such as Hank ' of 20~25 volumes of 0.2~2 volume
S balanced salt solutions, the Liberase MNP-S enzymes of 0.3~0.5 volume, the DNA I type enzymes of 0.5~1 volume, such as 22 volumes
Hank ' s balanced salt solutions, the Liberase MNP-S enzymes of 0.4 volume, the DNA I types enzyme of 0.7 volume).
4. method according to claim 1, the formula of the frozen stock solution is：65% DMEM-F12,15% human seralbumin egg
(HSA), 20% DMSO in vain.
5. method according to claim 1, the complete medium is the DMEM-F12 culture mediums for including 10%FBS.
6. method according to claim 1, wherein also including：
At least one of (7) placenta mesenchyma stem cell obtained by step (6), detection following items：Cytoactive, cell are dirty
Dye, hereditary disease, HLA-ABC/DR distribution type；Or, wherein also including：
(8) each after passing on obtained by step (6) freezes for placenta mesenchyma stem cell in liquid nitrogen；Or, wherein also including：
(9) database of the placenta stem-cell comprising information above is set up, and enters the database and the freeze-stored cell of step (8)
Row association.
7. method according to claim 1, wherein Hank's balanced salt solutions composition is：8.0g/L NaCl, 0.4g/L
KCl, 0.1g/L MgSO47H2O, 0.1g/L MgCl26H2O, 0.06g/L Na2HPO42H2O, 0.06g/L
KH2PO4,1.0g/L glucose, the phenol red of 0.14g/L CaCl2,0.35g/L NaHCO3,0.2g/L, hydrochloric acid or hydrogen
Sodium oxide molybdena adjusts pH to 7.4；For example, in wherein described mixed enzyme digestive juice except comprising Hank's balanced salt solutions,
Outside Liberase MNP-S enzymes, DNA I type enzymes, also added with zinc chloride.
8. method according to claim 1, wherein：
The cytoactive detection is that the number for freezing front and rear living cells is counted using trypan blue staining；
The cell contamination detection utilizes a small amount of cell culture, and whether detection cell is polluted by fungi and bacterium；For example, institute
It is to utilize aetology method to state cell contamination detection, and whether detection cell is by selected from one or more following of infection：Second
Double of liver two, hepatitis, AIDS virus, cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb,
HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, TRUST；
The hereditary disease detection is the method using molecular genetics, and detection freeze-stored cell whether there is hereditary disease；And/or
The HLA-ABC/DR distribution type is detection cell HLA-ABC/DR phenotypes.
9. method according to claim 1, wherein the placenta mesenchyma stem cell is to be frozen through program temperature-fall period in liquid nitrogen
In.
10. method according to claim 1, wherein the database include to all related data of the cell preserved,
Including but not limited to：The biological characteristics testing result of cell, multi-lineage potential qualification result, cellular elements genetic diagnosis
As a result, the particulars of fetus and its father and mother.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101270349A (en) * | 2008-03-20 | 2008-09-24 | 浙江大学 | Method for placenta mesenchyma stem cell separation and in vitro amplify cultivation |
| CN102586184A (en) * | 2011-09-05 | 2012-07-18 | 博雅干细胞科技有限公司 | Method for establishing placental mesenchyme stem cell library |
| CN102660501A (en) * | 2012-05-21 | 2012-09-12 | 博雅干细胞科技有限公司 | Method for separating and amplifying mesenchymal stem cell from fresh tissue of umbilical cord |
| CN102676451A (en) * | 2011-09-05 | 2012-09-19 | 博雅干细胞科技有限公司 | Method for separating mesenchymal stem cells from placenta |
| CN102807966A (en) * | 2012-08-16 | 2012-12-05 | 博雅干细胞科技有限公司 | Method for freezing and thawing placental whole cells and separating and expanding stem cells |
| CN105002136A (en) * | 2015-07-31 | 2015-10-28 | 夏凡 | Separation method of human placenta mesenchymal stem cells |
| CN105018421A (en) * | 2015-05-27 | 2015-11-04 | 贵州北科泛特尔生物科技有限公司 | Culturing method for human placental mesenchymal stem cells |
| CN106754674A (en) * | 2016-12-09 | 2017-05-31 | 博雅干细胞科技有限公司 | Method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion |
-
2017
- 2017-08-02 CN CN201710653583.1A patent/CN107299082B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101270349A (en) * | 2008-03-20 | 2008-09-24 | 浙江大学 | Method for placenta mesenchyma stem cell separation and in vitro amplify cultivation |
| CN102586184A (en) * | 2011-09-05 | 2012-07-18 | 博雅干细胞科技有限公司 | Method for establishing placental mesenchyme stem cell library |
| CN102676451A (en) * | 2011-09-05 | 2012-09-19 | 博雅干细胞科技有限公司 | Method for separating mesenchymal stem cells from placenta |
| CN102660501A (en) * | 2012-05-21 | 2012-09-12 | 博雅干细胞科技有限公司 | Method for separating and amplifying mesenchymal stem cell from fresh tissue of umbilical cord |
| CN102807966A (en) * | 2012-08-16 | 2012-12-05 | 博雅干细胞科技有限公司 | Method for freezing and thawing placental whole cells and separating and expanding stem cells |
| CN105018421A (en) * | 2015-05-27 | 2015-11-04 | 贵州北科泛特尔生物科技有限公司 | Culturing method for human placental mesenchymal stem cells |
| CN105002136A (en) * | 2015-07-31 | 2015-10-28 | 夏凡 | Separation method of human placenta mesenchymal stem cells |
| CN106754674A (en) * | 2016-12-09 | 2017-05-31 | 博雅干细胞科技有限公司 | Method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion |
Non-Patent Citations (3)
| Title |
|---|
| LEELANKFORD ET AL: "Manufacture and preparation of human placenta-derived mesenchymal stromal cells for local tissue delivery", 《CYTOTHERAPY》 * |
| N. V. NIZYAEVA ET AL: "Conditions for Collection of Placental Tissue Samples for Culturing of Multipotent Mesenchymal Stromal Cells Culturing of Multipotent Mesenchymal Stromal Cells", 《BULLETIN OF EXPERIMENTAL BIOLOGY AND MEDICINE》 * |
| 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》 * |
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| CN109699634A (en) * | 2019-01-31 | 2019-05-03 | 和携科技(北京)有限公司 | A kind of Cryopreservation and method for resuscitation of mescenchymal stem cell |
| CN110499287A (en) * | 2019-08-30 | 2019-11-26 | 博雅干细胞科技有限公司 | The method for simply preparing placenta mesenchyma stem cell excretion body |
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