CN108998409A - The isolation and purification method of Human embryo trophocyte and placenta mesenchyma stem cell - Google Patents
The isolation and purification method of Human embryo trophocyte and placenta mesenchyma stem cell Download PDFInfo
- Publication number
- CN108998409A CN108998409A CN201810904418.3A CN201810904418A CN108998409A CN 108998409 A CN108998409 A CN 108998409A CN 201810904418 A CN201810904418 A CN 201810904418A CN 108998409 A CN108998409 A CN 108998409A
- Authority
- CN
- China
- Prior art keywords
- stem cell
- placenta
- mesenchyma stem
- human embryo
- placenta mesenchyma
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000002826 placenta Anatomy 0.000 title claims abstract description 67
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 67
- 210000003716 mesoderm Anatomy 0.000 title claims abstract description 56
- 210000001161 mammalian embryo Anatomy 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 34
- 238000002955 isolation Methods 0.000 title claims abstract description 18
- 238000000746 purification Methods 0.000 title claims abstract description 17
- 210000004027 cell Anatomy 0.000 claims abstract description 70
- 229920001661 Chitosan Polymers 0.000 claims abstract description 50
- 239000012528 membrane Substances 0.000 claims abstract description 43
- 239000006285 cell suspension Substances 0.000 claims abstract description 29
- 238000000926 separation method Methods 0.000 claims abstract description 20
- 239000003480 eluent Substances 0.000 claims abstract description 15
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 12
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 12
- 238000003795 desorption Methods 0.000 claims abstract description 11
- 239000007788 liquid Substances 0.000 claims description 30
- 239000011521 glass Substances 0.000 claims description 20
- 238000001179 sorption measurement Methods 0.000 claims description 11
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 238000010790 dilution Methods 0.000 claims description 8
- 239000012895 dilution Substances 0.000 claims description 8
- 238000011010 flushing procedure Methods 0.000 claims description 8
- 210000005059 placental tissue Anatomy 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 229920001917 Ficoll Polymers 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 239000002159 nanocrystal Substances 0.000 claims description 5
- 239000000243 solution Substances 0.000 claims description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 230000001413 cellular effect Effects 0.000 claims description 4
- 238000007334 copolymerization reaction Methods 0.000 claims description 4
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 claims description 3
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 3
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000005520 cutting process Methods 0.000 claims description 3
- 230000029087 digestion Effects 0.000 claims description 3
- 230000001079 digestive effect Effects 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 235000019441 ethanol Nutrition 0.000 claims description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- MIKKOBKEXMRYFQ-WZTVWXICSA-N meglumine amidotrizoate Chemical compound C[NH2+]C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO.CC(=O)NC1=C(I)C(NC(C)=O)=C(I)C(C([O-])=O)=C1I MIKKOBKEXMRYFQ-WZTVWXICSA-N 0.000 claims description 3
- QNILTEGFHQSKFF-UHFFFAOYSA-N n-propan-2-ylprop-2-enamide Chemical compound CC(C)NC(=O)C=C QNILTEGFHQSKFF-UHFFFAOYSA-N 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- 239000002504 physiological saline solution Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- 238000001291 vacuum drying Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- AXAVXPMQTGXXJZ-UHFFFAOYSA-N 2-aminoacetic acid;2-amino-2-(hydroxymethyl)propane-1,3-diol Chemical compound NCC(O)=O.OCC(N)(CO)CO AXAVXPMQTGXXJZ-UHFFFAOYSA-N 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 229920001577 copolymer Polymers 0.000 claims description 2
- 239000000835 fiber Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 238000004064 recycling Methods 0.000 claims 2
- 230000015572 biosynthetic process Effects 0.000 claims 1
- 210000000630 fibrocyte Anatomy 0.000 claims 1
- 230000009870 specific binding Effects 0.000 abstract description 5
- 230000004083 survival effect Effects 0.000 abstract description 5
- 238000001816 cooling Methods 0.000 abstract description 2
- 230000003247 decreasing effect Effects 0.000 abstract description 2
- 235000018102 proteins Nutrition 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 8
- 208000027418 Wounds and injury Diseases 0.000 description 5
- 239000011324 bead Substances 0.000 description 5
- 230000027455 binding Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 102100032912 CD44 antigen Human genes 0.000 description 4
- 102100037241 Endoglin Human genes 0.000 description 4
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 4
- 101000881679 Homo sapiens Endoglin Proteins 0.000 description 4
- 101000935043 Homo sapiens Integrin beta-1 Proteins 0.000 description 4
- 102100025304 Integrin beta-1 Human genes 0.000 description 4
- 230000001464 adherent effect Effects 0.000 description 4
- 229920002678 cellulose Polymers 0.000 description 4
- 239000001913 cellulose Substances 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 210000002950 fibroblast Anatomy 0.000 description 4
- 125000001176 L-lysyl group Chemical class [H]N([H])[C@]([H])(C(=O)[*])C([H])([H])C([H])([H])C([H])([H])C(N([H])[H])([H])[H] 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 230000022558 protein metabolic process Effects 0.000 description 3
- 150000003254 radicals Chemical class 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 206010052428 Wound Diseases 0.000 description 2
- 230000005779 cell damage Effects 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 208000003142 Retained Placenta Diseases 0.000 description 1
- 206010038758 Retained placenta or membranes Diseases 0.000 description 1
- 101710109488 Salt stress-induced protein Proteins 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Gynecology & Obstetrics (AREA)
- Reproductive Health (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses the isolation and purification methods of a kind of Human embryo trophocyte and placenta mesenchyma stem cell, comprising: (1) placenta is handled；(2) cell suspension pre-separation；(3) streaming adsorbing separation；(4) desorption of placenta mesenchyma stem cell with separate；(5) purifying of Human embryo trophocyte.Antibody protein is planted on the outer surface of Thermo-sensitive chitosan copolymer membrane by the present invention, it is identified by surface molecular of the antibody to placenta mesenchyma stem cell, achieve the purpose that specific binding, and the endless form successively decreased with flow velocity remains in placenta mesenchyma stem cell on special Thermo-sensitive chitosan copolymer membrane.Recycle the mode of cooling plus eluent carry out placenta mesenchyma stem cell and the desorption of protein antibodies with separate.Separate mode of the invention is mild, and cell survival rate is high, and purity is also high.
Description
Technical field
The invention belongs to field of biomedicine technology, and in particular to Human embryo trophocyte and placenta mesenchyma stem cell
Isolation and purification method.
Background technique
Trophocyte cultivate and pass in vitro it is relatively difficult, Many researchers all using separation cell after directly processing or
The scheme of intervention, therefore disposably obtain a large amount of trophocyte and experiment is carried out to be very favorable.Placenta mesenchyma is dry thin
Born of the same parents are low in the abundance of placenta tissue, want that obtaining a certain number of primary cells is also required to many placenta tissues.
Using conventional method currently used for trophocyte and placenta mesenchyma stem cell includes following four:
(1) adherent partition method.The method mainly utilizes stem cell that there is the characteristic of adherent growth in plastic culture bottle will do
Cell is mutually separated with other cells, and this separation method separation cell is limited, and stem cell easily breaks up, thus influences its clinic
Application value.Moreover, contain adherent fibroblast simultaneously in isolated attached cell other than having stem cell, from
It is similar both in form not to be easily distinguishable.
(2) fluidic cell separating method.The method be it is small in size according to stem cell, lack less granular characteristic relatively it divided
From.
(3) immunomagnetic beads method.The method is that the magnetic bead and stem cell surface of antibody are had using packet according to immunology principle
Some distinctive marks such as CD34 specific bond, by being sorted when some strength magnetic field by delay.Using fluidic cell separating method
Or the stem cell purity that immunomagnetic beads obtain is larger, but obtained cell quantity is few, and easily causes cellular damage, cell
Survival rate is extremely low.
(4) density-gradient centrifugation method.The method is different from the density of other cells according to stem cell, experiments have shown that interstitial is dry
Cell is located at low-density cellular layer.Used common gradient separations liquid has Percoll and Ficoll, Ficoll method or Peroll
Method can effectively separate candidate stem cell from marrow, and purity is higher, and cell survival is good.But with Ficoll points
From cell in be often mixed with other cells (the non-mononuclearcell in such as fibroblast, marrow or blood).
Thermo-sensitive chitosan copolymer membrane due to making it under the premise of not needing enzymatic isolation method with good temperature-sensing property,
It only can achieve the purpose that cell absorption and the desorption of manual control by changing temperature, more be heated compared to immunomagnetic beads method
With, it is small for the damage of cell, and then cell success rate is high, is easier to disposably obtain a large amount of primary cells, by its by with spy
Heterogenetic antibody protein binding realizes that the research of the isolation and purification of cell is then rarely reported.
Summary of the invention
For the technical problem present on, the present invention provides a kind of Human embryo trophocyte and placenta mesenchyma stem cell
Isolation and purification method.
The technical solution of the present invention is as follows: a kind of side of isolating and purifying of Human embryo trophocyte and placenta mesenchyma stem cell
Method, comprising the following steps:
(1) placenta is handled: after placenta is carried out cleaning and bacteria removing, obtaining placenta tissue, then will implement placenta tissue
Tissue digestion is carried out using digestive juice, obtains a cell suspension；
(2) cell suspension pre-separation: a certain amount of cell suspension is taken to move in centrifuge tube, and into centrifuge tube
It is slowly added to cell separating liquid, the volume ratio of cell separating liquid and a cell suspension is 1:7-10, at 37 DEG C, 1300g centrifugation
Power, horizontal rotor are centrifuged 30min, take cellular layer, and cell is resuspended with PBS, obtain secondary cell suspension；Utilize first pre- point
From extracting the cell mixing layer of Human embryo trophocyte and placenta mesenchyma stem cell, exclusive PCR object；
(3) streaming adsorbing separation: by the secondary cell suspension using after diluted, machine is recycled to draw dilution
Secondary cell suspension afterwards is passed through total filled with special Thermo-sensitive chitosan at a temperature of 37-40 DEG C with certain initial flow rate
The glass tube of poly film flows out the secondary cell suspension of the glass tube again after machine is drawn, to be deteriorated speed in glass tube
Circulation 25min, then with dilution rinse glass tube, and phegma is collected, placenta mesenchyma stem cell specific adsorption exists
On special Thermo-sensitive chitosan copolymer membrane, Human embryo trophocyte is rested in the phegma；
(4) desorption of placenta mesenchyma stem cell with separate: to the special Thermo-sensitive for being adsorbed with placenta mesenchyma stem cell
Eluent is injected in chitosan copolymer membrane glass tube, and is cooled to 18-20 DEG C, closing stops 3-5min, then repeatedly with eluent
It rinses 5-8 times, collects flushing liquor, by the flushing liquor in 1000g centrifugal force, horizontal rotor is centrifuged 15min, supernatant is abandoned, then
PBS liquid is added into placenta mesenchyma stem cell to be resuspended, obtains primary placenta mesenchyma stem cell and counts；
(5) purifying of Human embryo trophocyte: by the phegma containing Human embryo trophocyte in 1000g centrifugal force, water
Flat turn is centrifuged 15min, abandons supernatant, then PBS liquid is added into Human embryo trophocyte and is resuspended, and is gone with differential attachment method
Except being counted after fibroblast.
Further, cell separating liquid described in step (2) includes: 7.8-12.6% nano junction in percentage by weight
Crystalline cellulose, 5.3-10.4% ficoll, 5.3-10.4% cardiografin, 1.5-3.5% sodium chloride, surplus are PBS buffer solution.
Wherein nanocrystal cellulose can absorb the harmful free radicals in cell suspension during centrifuge separation, reduce to cell
Damage.
Further, the density of the cell separating liquid is 1.077-1.079g/mL.
Further, in step (3) special Thermo-sensitive chitosan copolymer membrane preparation method the following steps are included:
(A) the Thermo-sensitive chitosan copolymer membrane made of acrylic acid, chitosan, n-isopropyl acrylamide raw material is chosen,
And it is cut to the size of 4 × 20cm；
(B) the Thermo-sensitive chitosan copolymer membrane after cutting is immersed in the CTAB solution of 0.1mM and handles 30min, make temperature
Quick property chitosan copolymerization film surface forms the spherical pit of dispersed, for increasing the specific surface of Thermo-sensitive chitosan copolymer membrane
Product, while improving its surface adhesion rate.Recycle aseptic water washing to still, vacuum drying, for use；
(C) antibody protein according to 5 × 106The Thermo-sensitive chitosan copolymer membrane of the density inoculation of a/mL after the drying
Outer surface is added confining liquid, is sealed to get special Thermo-sensitive chitosan copolymer membrane.It is dry to placenta mesenchyma by antibody
The surface molecular of cell is identified, to achieve the purpose that specific binding, placenta mesenchyma stem cell is remained in spy with this
On different Thermo-sensitive chitosan copolymer membrane.Wherein, any one in antibody protein CD29, CD44, CD105.
Further, in step (3) streaming adsorbing separation flow parameters are as follows: 0min, 10m/s；1-3min, 8m/
s；4-8min, 6m/s；9-14min, 4m/s；15-20min, 2m/s；21-25min, 1m/s.It will be grown containing Human embryo
It is poly- to flow successively through special Thermo-sensitive shell in such a way that flow velocity successively decreases for the cell mixing liquid for supporting cell and placenta mesenchyma stem cell
The joint efficiency of binding site and placenta mesenchyma stem cell on special Thermo-sensitive chitosan copolymer membrane can be improved in sugared copolymer membrane.
Because cycle-index is more, the quantity of binding site is fewer on special Thermo-sensitive chitosan copolymer membrane, if flow velocity is excessively high, no
Conducive to the absorption of a small amount of remaining placenta mesenchyma stem cell, while can also be carried out specifically since mistake high flow rate wash away part
Property combine placenta mesenchyma stem cell.
Further, the special Thermo-sensitive chitosan copolymer membrane is filled in the glass tube after wound, wound
It places compared to being only laid in glass inside pipe wall, placenta mesenchyma stem cell can be greatly increased and special Thermo-sensitive chitosan is total
The surface contact rate of poly film.
Further, dilution described in step (3) is physiological saline, PBS liquid, any one in Tris- glycine liquid
Or it is several.
Further, eluent described in step (4) is formed according to weight percent: 13.9-16.7% ethyl alcohol, 4.6-
5.2% phosphate, 5.4-6.1% triethylamine, 3.6-4.8%L- lysine salt, surplus are distilled water.Wherein, L-lysine salt
Protein metabolism in cell can be made to keep stablizing, to improve cell activity, increase the mildness of eluent.
Further, the temperature parameter in step (3) is preferably 37.5-38.5 DEG C, and it is living that excessively high temperature can destroy cell
Property, it is too low, lower adherence rate.
Further, the temperature parameter in step (4) is preferably 18.5-19.5 DEG C, too high or too low for temperature, be will cause
It is lower that efficiency is desorbed.
Compared with prior art, the invention has the benefit that
(1) present invention separates a cell suspension using cell separating liquid, can efficiently separate out Human embryo nourishing
The cell mixing layer of cell and placenta mesenchyma stem cell, exclusive PCR object, and the nanocrystal fiber in cell separating liquid
Element can absorb the harmful free radicals in cell suspension during centrifuge separation, reduce the damage to cell.
(2) antibody protein is planted on the outer surface of Thermo-sensitive chitosan copolymer membrane by the present invention, by antibody to placenta
The surface molecular of mescenchymal stem cell is identified, to achieve the purpose that specific binding, and the endless form successively decreased with flow velocity
Placenta mesenchyma stem cell is remained on special Thermo-sensitive chitosan copolymer membrane.It is milder compared to immunomagnetic beads screening,
Cell survival rate is higher.
(3) the characteristics of present invention is according to Thermo-sensitive chitosan copolymer membrane is incorporated between placenta and is filled by way of cooling
Matter stem cell is desorbed with protein antibodies, and is separated placenta mesenchyma stem cell with protein antibodies using eluent,
Wherein, the L-lysine salt in eluent can make protein metabolism in cell keep stablizing, to improve cell activity, increase
The mildness of eluent.
The present invention has separate mode mild, and cell survival rate is high, and then improves the purifying rate of cell.
Specific embodiment
Technical solution of the present invention is described in further detail below with reference to embodiment, but the present invention is not limited to following
The specific examples of act.
Embodiment 1
A kind of isolation and purification method of Human embryo trophocyte and placenta mesenchyma stem cell, comprising the following steps:
(1) placenta is handled: after placenta is carried out cleaning and bacteria removing, obtaining placenta tissue, then will implement placenta tissue
Tissue digestion is carried out using digestive juice, obtains a cell suspension；
(2) cell suspension pre-separation: a certain amount of cell suspension is taken to move in centrifuge tube, and into centrifuge tube
It is slowly added to cell separating liquid, the volume ratio of cell separating liquid and a cell suspension is 1:9, at 37 DEG C, 1300g centrifugal force,
Horizontal rotor is centrifuged 30min, takes cellular layer, and cell is resuspended with PBS, obtains secondary cell suspension；Using first pre-separation, mention
Take out the cell mixing layer of Human embryo trophocyte and placenta mesenchyma stem cell, exclusive PCR object；The cell separating liquid is pressed
It include: 11.5% nanocrystal cellulose, 8.4% ficoll, 7.9% cardiografin, 2.3% chlorination according to weight percent meter
Sodium, surplus are PBS buffer solution.Wherein nanocrystal cellulose can absorb the nocuousness in cell suspension during centrifuge separation
Free radical reduces the damage to cell.The density of the cell separating liquid is 1.078g/mL.
(3) streaming adsorbing separation: after the secondary cell suspension is diluted using dilution (physiological saline), machine is recycled
Device draws the secondary cell suspension after dilution, at a temperature of 37 DEG C, with certain initial flow rate by being filled with special Thermo-sensitive
The glass tube of chitosan copolymer membrane, special Thermo-sensitive chitosan copolymer membrane are attached on glass inside pipe wall, flow out the glass tube
Secondary cell suspension lead to 25min to be deteriorated speed in glass tube inner recirculation flow again after machine is drawn, then with dilution rinse
Glass tube, and phegma is collected, placenta mesenchyma stem cell specific adsorption is on special Thermo-sensitive chitosan copolymer membrane, people's embryo
Tire trophocyte rests in the phegma；The flow parameters of streaming adsorbing separation are as follows: 0min, 10m/s；1-3min,
8m/s；4-8min, 6m/s；9-14min, 4m/s；15-20min, 2m/s；21-25min, 1m/s.Someone's embryo will be contained
The cell mixing liquid of tire trophocyte and placenta mesenchyma stem cell, flows successively through special Thermo-sensitive in such a way that flow velocity successively decreases
The combination effect of binding site and placenta mesenchyma stem cell on special Thermo-sensitive chitosan copolymer membrane can be improved in chitosan copolymer membrane
Rate.Because cycle-index is more, the quantity of binding site is fewer on special Thermo-sensitive chitosan copolymer membrane, if flow velocity is excessively high,
Then it is unfavorable for the absorption of a small amount of remaining placenta mesenchyma stem cell, while can have also been carried out since mistake high flow rate washes away part
The placenta mesenchyma stem cell of specific binding.
The preparation method of special Thermo-sensitive chitosan copolymer membrane the following steps are included:
(A) the Thermo-sensitive chitosan copolymer membrane made of acrylic acid, chitosan, n-isopropyl acrylamide raw material is chosen,
And it is cut to the size of 4 × 20cm；
(B) the Thermo-sensitive chitosan copolymer membrane after cutting is immersed in the CTAB solution of 0.1mM and handles 30min, make temperature
Quick property chitosan copolymerization film surface forms the spherical pit of dispersed, for increasing the specific surface of Thermo-sensitive chitosan copolymer membrane
Product, while improving its surface adhesion rate.Recycle aseptic water washing to still, vacuum drying, for use；
(C) CD29 antibody protein according to 5 × 106The Thermo-sensitive chitosan copolymerization of the density inoculation of a/mL after the drying
The outer surface of film is added confining liquid, is sealed to get special Thermo-sensitive chitosan copolymer membrane.It is filled by antibody between placenta
The surface molecular of matter stem cell is identified, to achieve the purpose that specific binding, is retained placenta mesenchyma stem cell with this
On special Thermo-sensitive chitosan copolymer membrane.
(4) desorption of placenta mesenchyma stem cell with separate: to the special Thermo-sensitive for being adsorbed with placenta mesenchyma stem cell
Eluent is injected in chitosan copolymer membrane glass tube, and is cooled to 18 DEG C, closing stops 5min, then with eluent repeated flushing 8
It is secondary, flushing liquor is collected, by the flushing liquor in 1000g centrifugal force, horizontal rotor is centrifuged 15min, abandons supernatant, then between placenta
PBS liquid is added in mesenchymal stem cells to be resuspended, obtains primary placenta mesenchyma stem cell and counts；The eluent is according to weight
Measure percentage composition: 14.8% ethyl alcohol, 4.9% phosphate, 5.6% triethylamine, 4.3%L- lysine salt, surplus are distilled water.
Wherein, L-lysine salt can make protein metabolism in cell keep stablizing, to improve cell activity, increase the temperature of eluent
And property.
(5) purifying of Human embryo trophocyte: by the phegma containing Human embryo trophocyte in 1000g centrifugal force, water
Flat turn is centrifuged 15min, abandons supernatant, then PBS liquid is added into Human embryo trophocyte and is resuspended, and is gone with differential attachment method
Except being counted after fibroblast.
Embodiment 2
The present embodiment is substantially the same manner as Example 1, the difference is that the special Thermo-sensitive chitosan copolymer membrane passes through
It is filled in after wound in the glass tube, wound is placed compared to being only laid in glass inside pipe wall, can be greatly increased between placenta
The surface contact rate of mesenchymal stem cells and special Thermo-sensitive chitosan copolymer membrane.It is experimentally confirmed, in the case where other conditions are identical,
The special Thermo-sensitive chitosan copolymer membrane of wound shape improves 18% compared to the adsorption rate of adherent discoplacenta mescenchymal stem cell.
Embodiment 3
The present embodiment is substantially the same manner as Example 2, the difference is that the antibody protein is CD44.
Embodiment 4
The present embodiment is substantially the same manner as Example 2, the difference is that the antibody protein is CD105.Result of study hair
Existing, tri- kinds of antibody proteins of CD29, CD44, CD105 are not much different to the Percentage bound of placenta mesenchyma stem cell, wherein the knot of CD29
Conjunction rate can be slightly above tri- to five percentage points of CD44, CD105.
Embodiment 5
Influence of the different temperatures parameter to placenta mesenchyma stem cell adsorption rate in research step (3).
It is reference with embodiment 2,4 control groups is set, every group setting three parallel, remaining condition is all the same, and observation is thin
Born of the same parents' distribution situation, cell combination rate is as shown in table 1 under the adsorption temp in embodiment 2 and 4 control group in step (3):
Table 1: adsorption temp and cell combination rate
| Group | Adsorption temp | Cell combination rate |
| Embodiment 2 | 37℃ | 93.3% |
| Comparative example 1 | 36.5℃ | 85.2% |
| Comparative example 2 | 38.2℃ | 99.4% |
| Comparative example 3 | 40℃ | 94.6% |
| Comparative example 4 | 40.5℃ | 75.3% |
As shown in Table 1, adsorption temp is at 37-40 DEG C, and cell combination rate is 90% or more, and at 36.5 DEG C and 40.5
DEG C when cell combination rate have and decline by a relatively large margin it is too low because excessively high temperature can destroy cell activity, lower adherence rate.By
, it is found that adsorption temp is at 38.2 DEG C, cell combination rate is up to 99.4% for this.
Embodiment 6
Influence of the different temperatures parameter to placenta mesenchyma stem cell adsorption rate in research step (4).
It is reference with embodiment 2,4 control groups is set, every group setting three parallel, remaining condition is all the same, and observation is thin
Born of the same parents' distribution situation, Cell detachment rate is as shown in table 2 under the desorption temperature in embodiment 2 and 4 control group in step (3):
Table 2: desorption temperature and Cell detachment rate
| Group | Desorption temperature | Cell detachment rate |
| Embodiment 2 | 18℃ | 93.1% |
| Comparative example 1 | 17.5℃ | 89.3% |
| Comparative example 2 | 19.3℃ | 99.3% |
| Comparative example 3 | 20℃ | 95.8% |
| Comparative example 4 | 21℃ | 80.3% |
As shown in Table 2, at 18-20 DEG C, Cell detachment rate has small desorption temperature 93% or more at 17.5 DEG C
Amplitude decline, and have sharp fall at 21 DEG C, it follows that desorption temperature, at 19.3 DEG C, Cell detachment rate is up to
99.3%.
The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.
Claims (10)
1. a kind of isolation and purification method of Human embryo trophocyte and placenta mesenchyma stem cell, which is characterized in that including following
Step:
(1) placenta is handled: after placenta is carried out cleaning and bacteria removing, obtaining placenta tissue, then the placenta tissue is utilized
Digestive juice carries out tissue digestion, obtains a cell suspension；
(2) cell suspension pre-separation: taking a certain amount of cell suspension to move in centrifuge tube, and slowly into centrifuge tube
Cell separating liquid is added, the volume ratio of cell separating liquid and a cell suspension is 1:7-10, at 37 DEG C, 1300g centrifugal force, water
Flat turn is centrifuged 30min, takes cellular layer, and cell is resuspended with PBS, obtains secondary cell suspension；
(3) streaming adsorbing separation: after the secondary cell suspension is diluted using after diluted, recycling machine is drawn
Secondary cell suspension, at a temperature of 37-40 DEG C, with certain initial flow rate by being filled with special Thermo-sensitive chitosan copolymer membrane
Glass tube, flow out the secondary cell suspension of the glass tube again after machine is drawn, speed recycles in glass tube to be deteriorated
Circulate 25min, then with dilution rinse glass tube, and collects phegma, and placenta mesenchyma stem cell specific adsorption is special
On Thermo-sensitive chitosan copolymer membrane, Human embryo trophocyte is rested in the phegma；
(4) desorption of placenta mesenchyma stem cell with separate: it is poly- to the special Thermo-sensitive shell for being adsorbed with placenta mesenchyma stem cell
Eluent is injected in sugared copolymer membrane glass tube, and is cooled to 18-20 DEG C, closing stops 3-5min, then with eluent repeated flushing
5-8 times, flushing liquor is collected, by the flushing liquor in 1000g centrifugal force, horizontal rotor is centrifuged 15min, abandons supernatant, then to tire
PBS liquid is added in disk mescenchymal stem cell to be resuspended, obtains primary placenta mesenchyma stem cell and counts；
(5) purifying of Human embryo trophocyte: by the phegma containing Human embryo trophocyte in 1000g centrifugal force, level turns
Son centrifugation 15min, abandon supernatant, then into Human embryo trophocyte be added PBS liquid be resuspended, with differential attachment method removal at
It is counted after fibrocyte.
2. the isolation and purification method of a kind of Human embryo trophocyte and placenta mesenchyma stem cell according to claim 1,
It is characterized in that, cell separating liquid described in step (2) includes: 7.8-12.6% nanocrystal fiber in percentage by weight
Element, 5.3-10.4% ficoll, 5.3-10.4% cardiografin, 1.5-3.5% sodium chloride, surplus is PBS buffer solution.
3. the isolation and purification method of a kind of Human embryo trophocyte and placenta mesenchyma stem cell according to claim 2,
It is characterized in that, the density of the cell separating liquid is 1.077-1.079g/mL.
4. the isolation and purification method of a kind of Human embryo trophocyte and placenta mesenchyma stem cell according to claim 1,
It is characterized in that, in step (3) special Thermo-sensitive chitosan copolymer membrane preparation method the following steps are included:
(A) the Thermo-sensitive chitosan copolymer membrane made of acrylic acid, chitosan, n-isopropyl acrylamide raw material is chosen, is gone forward side by side
Row is cut；
(B) the Thermo-sensitive chitosan copolymer membrane after cutting is immersed in the CTAB solution of 0.1mM and handles 30min, make Thermo-sensitive
The spherical pit of chitosan copolymerization film surface formation dispersed, recycling aseptic water washing to still, vacuum drying, for use；
(C) antibody protein according to 5 × 106The appearance of the Thermo-sensitive chitosan copolymer membrane of the density inoculation of a/mL after the drying
Face is added confining liquid, is sealed to get special Thermo-sensitive chitosan copolymer membrane.
5. the isolation and purification method of a kind of Human embryo trophocyte and placenta mesenchyma stem cell according to claim 1,
It is characterized in that, in step (3) streaming adsorbing separation flow parameters are as follows: 0min, 10m/s；1-3min, 8m/s；4-
8min, 6m/s；9-14min, 4m/s；15-20min, 2m/s；21-25min, 1m/s.
6. the isolation and purification method of a kind of Human embryo trophocyte and placenta mesenchyma stem cell according to claim 1,
It is characterized in that, in step (3) streaming adsorbing separation flow parameters are as follows: 0min, 10m/s；1-3min, 8m/s；4-
8min, 6m/s；9-14min, 4m/s；15-20min, 2m/s；21-25min, 1m/s.
7. the isolation and purification method of a kind of Human embryo trophocyte and placenta mesenchyma stem cell according to claim 1,
It is characterized in that, dilution described in step (3) is physiological saline, any one or a few in PBS liquid, Tris- glycine liquid.
8. the isolation and purification method of a kind of Human embryo trophocyte and placenta mesenchyma stem cell according to claim 1,
It is characterized in that, eluent described in step (4) consists of the following compositions in percentage by weight: 13.9-16.7% ethyl alcohol,
4.6-5.2% phosphate, 5.4-6.1% triethylamine, 3.6-4.8%L- lysine salt, surplus are distilled water.
9. the isolation and purification method of a kind of Human embryo trophocyte and placenta mesenchyma stem cell according to claim 1,
It is characterized in that, the temperature parameter in step (3) is 37.5-38.5 DEG C.
10. the isolation and purification method of a kind of Human embryo trophocyte and placenta mesenchyma stem cell according to claim 1,
It is characterized in that, the temperature parameter in step (4) is 18.5-19.5 DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810904418.3A CN108998409B (en) | 2018-08-09 | 2018-08-09 | The isolation and purification method of Human embryo trophocyte and placenta mesenchyma stem cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810904418.3A CN108998409B (en) | 2018-08-09 | 2018-08-09 | The isolation and purification method of Human embryo trophocyte and placenta mesenchyma stem cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108998409A true CN108998409A (en) | 2018-12-14 |
| CN108998409B CN108998409B (en) | 2019-07-26 |
Family
ID=64594754
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810904418.3A Expired - Fee Related CN108998409B (en) | 2018-08-09 | 2018-08-09 | The isolation and purification method of Human embryo trophocyte and placenta mesenchyma stem cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108998409B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060254963A1 (en) * | 2002-12-25 | 2006-11-16 | Toshiyuki Kanamori | Cell separation and collection apparatus and separation and collection method |
| CN102516473A (en) * | 2011-12-14 | 2012-06-27 | 华南理工大学 | Copolymerized nano composite hydrogel for intelligent separation of cell sheets as well as preparation method and application thereof |
| CN102942651A (en) * | 2012-12-11 | 2013-02-27 | 天津工业大学 | Galactose-base temperature-sensitive microgel and preparation method thereof |
| US20140234890A1 (en) * | 2013-02-15 | 2014-08-21 | Douglas T. Gjerde | Methods for Purifying Biological Cells |
| CN105087470A (en) * | 2015-07-31 | 2015-11-25 | 何静 | Method for separating and purifying human embryo trophoblast and placental mesenchymal stem cells |
-
2018
- 2018-08-09 CN CN201810904418.3A patent/CN108998409B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060254963A1 (en) * | 2002-12-25 | 2006-11-16 | Toshiyuki Kanamori | Cell separation and collection apparatus and separation and collection method |
| CN102516473A (en) * | 2011-12-14 | 2012-06-27 | 华南理工大学 | Copolymerized nano composite hydrogel for intelligent separation of cell sheets as well as preparation method and application thereof |
| CN102942651A (en) * | 2012-12-11 | 2013-02-27 | 天津工业大学 | Galactose-base temperature-sensitive microgel and preparation method thereof |
| US20140234890A1 (en) * | 2013-02-15 | 2014-08-21 | Douglas T. Gjerde | Methods for Purifying Biological Cells |
| CN105087470A (en) * | 2015-07-31 | 2015-11-25 | 何静 | Method for separating and purifying human embryo trophoblast and placental mesenchymal stem cells |
Non-Patent Citations (4)
| Title |
|---|
| 冯霞等: "快速响应温敏凝胶及其在生物分离中的应用研究", 《南开大学学报(自然科学版)》 * |
| 常宪智: "壳聚糖交联温敏性水凝胶的合成和性质研究", 《中国优秀硕士学位论文全文数据库工程科技I辑》 * |
| 李媛媛等: "纳米纤维素纤维凝胶特性及其应用", 《纤维素科学与技术》 * |
| 贺晓凌等: "糖化温敏凝胶的制备及其与HepG2细胞相互作用研究", 《高分子学报》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108998409B (en) | 2019-07-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109097328A (en) | One species specific mescenchymal stem cell excretion body extracting method | |
| CN107988153A (en) | The method of mesenchymal stem cells derived from human umbilical blood source separation excretion body and the reagent used | |
| EP2322601A1 (en) | Method of collecting placental stem cells | |
| CN101638637B (en) | Kit for processing human marrow, cord blood and peripheral blood cells and cell processing method | |
| CN108184818B (en) | Human placenta mesenchymal stem cell suspension protective agent | |
| CN102735680B (en) | Test strip for quickly detecting porcine circovirus 2 (PCV2) antibody by adopting colloidal gold | |
| CN104630144A (en) | Method for separating and culturing umbilical cord blood mesenchymal stem cells | |
| CN102120768B (en) | Method for producing curative canine parvovirus virus monoclonal antibody by using bioreactor | |
| CN109251889A (en) | A kind of transplanting preparation system of dental pulp mescenchymal stem cell microballoon | |
| CN104711226A (en) | Preparation method of placenta hematopoietic stem cells | |
| CN108004206A (en) | It is a kind of from the preparation method of people's olfactory mucosa mescenchymal stem cell excretion body and the application of excretion body | |
| CN104762258B (en) | A kind of cultural method of human umbilical cord mesenchymal stem cells | |
| CN104130319A (en) | Extraction method of phycoerythrin | |
| CN109385396A (en) | Clinical application grade umbilical cord mesenchymal stem cells and its method for separating and preparing | |
| CN108865988A (en) | A kind of separation of human amnion mesenchymal stem cell, culture and purification process | |
| CN105601736B (en) | A kind of anti respiratory syncytial virus human immunoglobulin(HIg) and preparation method thereof | |
| CN104974980B (en) | A kind of separation method of human amniotic membrane epithelial cell | |
| CN106635769B (en) | Cell separation apparatus and cell isolation method | |
| CN108998409B (en) | The isolation and purification method of Human embryo trophocyte and placenta mesenchyma stem cell | |
| CN106702499A (en) | Canine mesenchymal stem cell bank and construction method thereof | |
| CN102250839A (en) | Universal cell processing kit and application method thereof | |
| CN107354130A (en) | A kind of intermembranous mesenchymal stem cells separation method of human placenia | |
| CN106635974A (en) | Method for separating and culturing human umbilical cord mesenchymal stem cells | |
| CN112159796A (en) | Primary isolation method and application of human umbilical cord-derived mesenchymal stem cells | |
| CN114507642B (en) | Method for separating single cells of pericytes of animal nervous system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190726 |