CN108148806A - A kind of fast separating process of placental hematopoietic stem cell - Google Patents
A kind of fast separating process of placental hematopoietic stem cell Download PDFInfo
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Abstract
The invention discloses a kind of fast separating process of placental hematopoietic stem cell of candidate stem cell separation technology field.The specific steps are：Normal labor, in vitro placenta are fully immersed in buffer solution；Placental lobules is shredded, placental lobules is fully rinsed with buffer solution, collects flushing liquor；It is filtered with copper mesh, collects the liquid after filtering；Mononuclearcell is centrifuged using density-gradient centrifugation method, it is washed with buffer solution, suspended obtained cell with candidate stem cell culture medium later, up to placental hematopoietic stem cell, the placenta source hemopoietic stem cell has the biological function identical with Cord Blood-Derived candidate stem cell, and the candidate stem cell quantity obtained can meet needed for adult's transplanting.Separation method of the present invention is easy to operate, convenient and practical；Placenta cells composition is inmatureer, derives from a wealth of sources, and is conveniently easy to get, and has broad prospects in the clinical practice of stem cell.
Description
Technical field
The invention belongs to candidate stem cell separation technology fields, and in particular to a kind of quick separating of placental hematopoietic stem cell
Method.
Background technology
Candidate stem cell (Hematopoietic stem cell, HSC) is detached from marrow earliest to be obtained, and is come
Derived from mesoblastic a kind of adult stem cell with multi-lineage potential and self-renewal capacity, in vivo and external specific item
With the ability for hemopoietic system being respectively cell differentiation under part, mainly including medullary system, leaching system, macronucleus system etc..Newest research table
Bright, candidate stem cell not only plays a significant role in terms of body hemopoietic system and immune system is rebuild, but also utilizes gene work
Journey technological sourcing foreign gene carries out treating correlative diseases and has also obtained the effect of attracting people's attention.Therefore candidate stem cell will be in future
It has broad application prospects in terms of clinical treatment.
The candidate stem cell reported at present is mainly derived from marrow, Cord blood and mobilized peripheral blood, but above-mentioned three kinds are come
The candidate stem cell in source has certain limitation.The classical way that derived from bone marrow is obtained as hematopoietic stem cell transplantation seed cell
Diameter, a painful operation of comparison need to be undergone, and have very high infection machine during materials and after materials by taking the process of marrow
Rate；And with the increase at donor age, quantity, proliferation and the differentiation capability of candidate stem cell are remarkably decreased in marrow, make it
It is studying and is applying, be especially restricted in clinical practice.A kind of new sources of the Cord blood as candidate stem cell, have
Materials easily, the advantages that not limited by moral ethics, but due to candidate stem cell limited amount in Cord blood, be insufficient for into
Needed for people's transplanting, therefore Cord blood is only limited and is used in children.It is thin that although mobilized peripheral blood can obtain sufficient amount of Hematopoietic Stem
Born of the same parents transplant for being grown up, but its somewhat expensive, need to carry out donor the injection of mobilization agent to obtain transplanting Hematopoietic Stem used
Cell.Injecting mobilization agent in itself has donor certain damage, while also need to pay high mobilization agent expense, it is therefore desirable to seek
New derived from hematopoietic precursor cells is looked for, to meet needed for transplanting.
Placenta originating from embryonic development period extraembryonic mesoderm is made of interstitial, blood vessel and trophocyte, containing a large amount of
Blood cell composition.Our result of study shows to detach what is obtained from placenta containing abundant candidate stem cell in placenta
Candidate stem cell has the biological function identical with Cord blood, this will open up one for experimental study from now on and clinical practice
Brand-new and abundant source.
Patent of invention application No. is 201610570495.0 discloses a kind of placental hematopoietic stem cell extraction preparation method,
The specific steps are：Step 1, normal natural labor or the healthy placenta of caesarean birth person are acquired under sterile operating room environment, then with clear
Washing lotion carries out cleaning treatment early period to placenta, collects treated liquid A；Step 2, with irrigating solution difference lavation placenta navel it is quiet,
Endarterial blood collects the liquid B after lavation；Step 3, placenta is shredded, digests placenta tissue with enzymolysis liquid, collect enzymolysis
The liquid C obtained afterwards；Step 4, it by the liquid A of above-mentioned collection, liquid B and liquid C and for mixed liquor, is taken after mixed liquor centrifugation thin
Born of the same parents are precipitated；Step 5, by cell precipitation by obtaining the placental hematopoietic stem cell after resuspension.The preparation method mainly use from
The mode of placenta umbilical vein, artery perfusion, existence time is long in practical operation, accurately finds arteria umbilicalis and umbilical vein difficulty, filling
The shortcomings of flow liquid perfusion difficulty, easy blood coagulation；By the way of enzymic digestion, what is obtained is candidate stem cell and hematopoieticmicroenviron-ment
The cell mixing solution rather than simple candidate stem cell of stroma cell and mescenchymal stem cell etc., and easily in receptor
The complication such as fibrosis are formed in vivo.
Invention content：
In order to solve the above-mentioned technical problem the present invention, proposes a kind of fast separating process of placental hematopoietic stem cell.Specifically
Technical solution is as follows：
A kind of fast separating process of placental hematopoietic stem cell, includes the following steps：
(1) normal labor, in vitro placenta are fully immersed in buffer solution；
(2) placental lobules is shredded, placental lobules is fully rinsed with buffer solution, collects flushing liquor；
(3) above-mentioned flushing liquor is filtered, collects the cell suspension after filtering；
(4) above-mentioned cell suspension is centrifuged into mononuclearcell using density-gradient centrifugation method, is washed with buffer solution, it
Obtained cell is suspended to get placental hematopoietic stem cell with candidate stem cell culture medium afterwards.
The method being immersed in placenta in buffer solution in step (1) is：First placenta is fully immersed in buffer solution, room temperature
2-6h is impregnated, is taken out, is then placed in another fresh buffer and impregnates.
Above-mentioned buffer solution is PBS buffer solution.
Buffer solution contains 10% volume fetal calf serum in step (2).
Filtering uses the copper mesh of 180~220 mesh in step (3).
Before the operation for carrying out step (4), the cell suspension after filtering is centrifuged, takes cell precipitation, is buffered with PBS
Cell is resuspended in liquid.
Before the operation for carrying out step (4), the cell suspension after filtering is centrifuged under 800g~1000g rotating speeds 8min~
12min takes cell precipitation, and cell is resuspended with the PBS buffer solution of the fetal calf serum containing 10% volume
The concrete operations of density-gradient centrifugation method described in step (4) are：Cell suspension is pressed 1:1 volume ratio is added to leaching
In bar cell separating liquid, 800g~1000g is centrifuged 20~40 minutes, collects intermediate tunica albuginea layer, and PBS buffer solution is washed 1~3 time,
400g~600g is centrifuged 8~15 minutes, and it is aim cell to collect mononuclearcell.
Candidate stem cell culture medium described in step (4) is the IMDM culture mediums containing 10% volume fetal calf serum.
Gained placental hematopoietic stem cell is frozen using Programmed cryopreservation instrument in step (4), is then transferred in liquid nitrogen
It preserves.
Beneficial effects of the present invention are：
(1) preparation of existing placental hematopoietic stem cell is main using by the way of placenta umbilical vein, artery perfusion, and reality is grasped
Existence time length, accurate the shortcomings of finding arteria umbilicalis and umbilical vein difficulty, perfusion perfusion difficulty, easy blood coagulation in work；And this
Invention is detached from placenta using crush method combination perfusion wash method and obtains placental hematopoietic stem cell, easy to operate.
(2) placenta is shredded in the prior art, digests placenta tissue with enzymolysis liquid, what is obtained is candidate stem cell and hematopoiesis
The cell mixing solution rather than simple candidate stem cell of the stroma cell and mescenchymal stem cell of microenvironment etc., and face
The complication such as fibrosis are easily formed in recipient's body during bed application；And the present invention rinses placental lobules using PBS buffer solution, obtains
Largely simple candidate stem cell is obtained, which has the life identical with Cord Blood-Derived candidate stem cell
Object function has and to hematopoiesis is respectively cell differentiation and rebuilds hematopoietic potential, for future clinical treatment provide it is abundant it is dry carefully
Born of the same parents source, and the complication such as fibrosis are formed when avoiding clinical practice in recipient's body.
(3) separation method of the present invention is easy to operate, convenient and practical；And since placenta is as Cord blood, Cell Component compared with
Naivety derives from a wealth of sources, and is conveniently easy to get, and has broad prospects in the clinical practice of stem cell.
The placenta that (4) weight are 450-600 grams can averagely obtain 1 × 109~2 × 109Mononuclearcell, CD34+
Cell is 2 × 106~8 × 106A cell shows the biological characteristics with candidate stem cell after identification, cell quality is good, institute
The cell quantity obtained can meet needed for adult's transplanting.
Description of the drawings
Fig. 1 is the morphological observation under microscope；Wherein, A is the mononuclearcell that placental blood separation obtains；B is
The candidate stem cell of acquisition is detached under high power lens.
Fig. 2 identifies HSC surface marker results for flow cytometry.
Fig. 3 is placenta HSC amplification in vitro results.
Fig. 4 is the plastidogenetic hematopoietic colonies of placenta HSC.
Specific embodiment
Following embodiment facilitates a better understanding of the present invention.Experimental method is routine experiment method unless otherwise specified,
Experiment material can be obtained unless otherwise specified by commercial sources.
Embodiment 1：The separation method of placenta HSC
The placenta of 450-600g normal labors is taken under aseptic condition within postpartum 4-6 hour, and is immersed in PBS buffer solution
In, room temperature preserves to be handled as early as possible within 2-6h；Placenta from the PBS buffer solution of immersion is taken out in Biohazard Safety Equipment, is placed in another
In one aseptic operation pallet, add in the fresh PBS buffer solution of 800-1000mL and be completely soaked, and shredded completely with surgical scissors
Placental lobules fully rinses placental lobules with PBS buffer solution, washes out remaining blood in placental lobules, and collect rinse rapidly
Liquid；200 mesh copper mesh of flushing liquor are filtered, collect the cell suspension after filtering, and be added in multiple 50ml centrifuge tubes, 900g
Centrifugation 10 minutes, outwells supernatant, and cell is resuspended with the PBS buffer solution containing 10% volume fetal calf serum (FBS)；Then cell is hanged
Liquid presses 1:1 volume ratio is added in lymphocyte separation medium (Ficoll), and 900g is centrifuged 30 minutes, collects intermediate tunica albuginea layer, PBS
Buffer solution is washed 2 times, and each 500g is centrifuged 10 minutes, and it is aim cell to collect mononuclearcell, and PBS buffer solution washs single core
After cell, suspended obtained cell, as placental hematopoietic stem cell with the IMDM culture mediums containing 10% volume FBS.
The number for the living cells that separation obtains is counted with trypan blue staining：10ul placenta HSC suspensions are taken, by cell suspension
Trypan blue dye liquor with 0.4% presses 1:1 volume ratio mixing is drawn 10ul cell mixtures and is added in blood cell counting plate,
Living cell counting number under microscope.The placenta that one weight is 450-600 grams can averagely obtain 1 × 109-2×109A list
A nucleus.
Embodiment 2：Placenta HSC's freezes
After the candidate stem cell isolated and purified is carried out trypan blue counting, according to 2 × 106Cell is added to 1ml cells jelly
In liquid storage (culture solution containing 50%IMEM, 40%FBS, 10% dimethyl sulfoxide (DMSO)), liquid nitrogen is finally putting by Programmed cryopreservation
It is frozen in pipe.
Embodiment 3：The Identification of Biological Characteristics of placenta HSC
First, cytomorphology feature
By isolating and purifying for embodiment 1, placenta mononuclearcell under the microscope (scheme by visible rounded suspension cell
1-A), using being inverted ultramicroscopic observation candidate stem cell, it is seen that in the cell (Fig. 1-B) of typical round suspension growth.
2nd, flow cytometry identification placenta HSC surface markers
Take the cell that density gradient method isolates and purifies, Flow cytometry cell surface marker.Cell is collected, after counting
Take 8 × 106A cell, 8 pipe of packing；PBS buffer solution is washed once, 1500rpm centrifugations 10min；Supernatant is abandoned, remains 100~200 μ
L blows and beats mixing cell；Add in CD38, CD133 antibody (being purchased from BD companies) of PE labels and CD45, CD34 (purchase of FITC labels
From BD companies) each 10 μ l of antibody, and a pipe is set as blank control；4 DEG C are protected from light 30min；PBS buffer solution is washed once,
1500rpm centrifuges 10min；The cell directly marked abandons supernatant, adds in 200 μ l PBS buffer solution piping and druming mixing cell, 200 μ l's
1% paraformaldehyde is fixed, and puts 4 DEG C of to be measured, flow cytometer detections in 3 days.
Flow cytomery is the result shows that (Fig. 2), CD34 positive rates are for (0.38 ± 0.05) %, CD133 positive rate
(0.84 ± 0.36) %, gained positive cell are similar to umbilical hemopoietic stem cell.The placenta that one weight is 450-600 grams is averaged
CD34 can be obtained+Cell 2 × 106-8×106It is a.
3rd, placenta HSC amplification in vitros result
With addition 10-6The early stage candidate stem cell long-term cultivation base 5100 (Stem Cell Products) of M hydrocortisones
And/or cell factor cultivating system, combination of cytokines include SCF 50ng/ml, FL 50ng/ml, TPO 50ng/ml, inoculation
Placenta CD34+Cell is in 24 orifice plates, and 1 × 104Cells/well, per hole 1ml, 33 DEG C, 5%CO2Culture 12 days, half amount is changed within the 6th day
Liquid.Each group total number of cells, CD34 are detected respectively in 0d and 12d+Cell number, Colony forming.It is above-mentioned the experimental results showed that (figure
3) ability that the cell of acquisition has hematopoietic stem cell population, is detached, there are the biological characteristics of candidate stem cell.
4th, the Colony forming ability detection of placenta HSC
With the semifixed colonies culture detection placenta HSC Colony forming abilities of methylcellulose.Cultivating system is：IMDM is trained
It supports in base containing 30%HS, 1%BSA, 2mmol/L L- glutamine, 1 × 10-4Mol/L 2 mercapto ethanols, 1% methylcellulose
And recombinant cytokine.Cell factor (rhSCF 50ng/ml, rhIL-310ng/ml, rhGM-CSF10ng/ml, rhEPO 6U/
ml).With the Petri-dish culture dishes of 35mm at 37 DEG C, 5%CO2Colony cultivation is carried out under complete wet condition of culture；What is be inoculated with is thin
Born of the same parents' number is 1000 cells of 0d/cell/ml of ml, 12d 4000, and 2ml/ wares, three multiple ware/groups co-culture 12d, aobvious being inverted
It is directly counted under micro mirror, more than 50 cells are a colony, above-mentioned the experimental results showed that (Fig. 4), separated to purify what is obtained
Cell has the characteristic and activity of candidate stem cell.
Claims (10)
1. a kind of fast separating process of placental hematopoietic stem cell, which is characterized in that include the following steps：
(1) normal labor, in vitro placenta are fully immersed in buffer solution；
(2) placental lobules is shredded, placental lobules is fully rinsed with buffer solution, collects flushing liquor；
(3) above-mentioned flushing liquor is filtered, collects the cell suspension after filtering；
(4) above-mentioned cell suspension is centrifuged into mononuclearcell using density-gradient centrifugation method, is washed with buffer solution, Zhi Houyong
Candidate stem cell culture medium suspends obtained cell to get placental hematopoietic stem cell.
2. separation method according to claim 1, which is characterized in that placenta is immersed in buffer solution by step in (1)
Method is：First placenta is fully immersed in buffer solution, soak at room temperature 2-6h, takes out, be then placed in another fresh buffer
It impregnates.
3. separation method according to claim 1 or 2, which is characterized in that the buffer solution is PBS buffer solution.
4. separation method according to claim 1, which is characterized in that buffer solution contains 10% volume tire ox in step (2)
Serum.
5. separation method according to claim 1, which is characterized in that filtering uses the copper of 180~220 mesh in step (3)
Net.
6. separation method according to claim 1, which is characterized in that before the operation for carrying out step (4), after filtering
Cell suspension is centrifuged, and takes cell precipitation, and cell is resuspended with PBS buffer solution.
7. separation method according to claim 6, which is characterized in that before the operation for carrying out step (4), after filtering
Cell suspension centrifuges 8min~12min under 800g~1000g rotating speeds, takes cell precipitation, with the fetal calf serum containing 10% volume
PBS buffer solution be resuspended cell.
8. separation method according to claim 1, which is characterized in that the tool of density-gradient centrifugation method described in step (4)
Gymnastics conduct：Cell suspension is pressed 1:1 volume ratio is added in lymphocyte separation medium, and 800g~1000g centrifuges 20~40 points
Clock collects intermediate tunica albuginea layer, and PBS buffer solution is washed 1~3 time, and 400g~600g is centrifuged 8~15 minutes, collects mononuclearcell and is
For purpose cell.
9. separation method according to claim 1, which is characterized in that candidate stem cell culture medium is described in step (4)
IMDM culture mediums containing 10% volume fetal calf serum.
10. separation method according to claim 1, which is characterized in that gained placental hematopoietic stem cell uses in step (4)
Programmed cryopreservation instrument is frozen, and is then transferred in liquid nitrogen and is preserved.
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Cited By (3)
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| CN111269942A (en) * | 2020-02-28 | 2020-06-12 | 中国人民解放军军事科学院军事医学研究院 | Application of TGFBI as marker for regulating and controlling osteogenic differentiation of mesenchymal stem cells |
| CN111647551A (en) * | 2020-06-12 | 2020-09-11 | 银丰生物工程集团有限公司 | Method for extracting and preserving cells from same placenta villus lobule tissue |
| CN114591904A (en) * | 2022-03-09 | 2022-06-07 | 河南省组织细胞库有限公司 | Method for extracting natural killer cells from placenta |
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