CN109423476A - It is used to prepare the kit of fat mesenchymal stem cell - Google Patents
It is used to prepare the kit of fat mesenchymal stem cell Download PDFInfo
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- CN109423476A CN109423476A CN201710717884.6A CN201710717884A CN109423476A CN 109423476 A CN109423476 A CN 109423476A CN 201710717884 A CN201710717884 A CN 201710717884A CN 109423476 A CN109423476 A CN 109423476A
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- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 52
- 210000004027 cell Anatomy 0.000 claims abstract description 72
- 239000000243 solution Substances 0.000 claims abstract description 61
- 238000005406 washing Methods 0.000 claims abstract description 29
- 230000004130 lipolysis Effects 0.000 claims abstract description 17
- 239000007788 liquid Substances 0.000 claims description 52
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 claims description 39
- 229960001138 acetylsalicylic acid Drugs 0.000 claims description 39
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 claims description 38
- 229960003957 dexamethasone Drugs 0.000 claims description 38
- 239000012930 cell culture fluid Substances 0.000 claims description 24
- 238000004140 cleaning Methods 0.000 claims description 23
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 21
- 239000001963 growth medium Substances 0.000 claims description 19
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 18
- 239000000872 buffer Substances 0.000 claims description 15
- 238000010494 dissociation reaction Methods 0.000 claims description 15
- 230000005593 dissociations Effects 0.000 claims description 15
- 229930182555 Penicillin Natural products 0.000 claims description 14
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 14
- 229940049954 penicillin Drugs 0.000 claims description 14
- 238000004321 preservation Methods 0.000 claims description 12
- 239000012981 Hank's balanced salt solution Substances 0.000 claims description 8
- 239000002504 physiological saline solution Substances 0.000 claims description 7
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 claims description 5
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 2
- 229930182816 L-glutamine Natural products 0.000 claims description 2
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 claims description 2
- 230000003115 biocidal effect Effects 0.000 claims description 2
- 230000001079 digestive effect Effects 0.000 claims description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 2
- 229960003531 phenolsulfonphthalein Drugs 0.000 claims description 2
- 239000011550 stock solution Substances 0.000 claims description 2
- 238000003860 storage Methods 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 12
- 230000029087 digestion Effects 0.000 abstract description 5
- 238000005138 cryopreservation Methods 0.000 abstract description 3
- 238000004113 cell culture Methods 0.000 abstract 1
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- 238000000034 method Methods 0.000 description 47
- 230000001954 sterilising effect Effects 0.000 description 26
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- 210000000130 stem cell Anatomy 0.000 description 22
- 238000002360 preparation method Methods 0.000 description 17
- 230000000694 effects Effects 0.000 description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 239000000203 mixture Substances 0.000 description 10
- 239000007640 basal medium Substances 0.000 description 9
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 6
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 5
- 102000004142 Trypsin Human genes 0.000 description 5
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- 238000001514 detection method Methods 0.000 description 5
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 5
- 229910000397 disodium phosphate Inorganic materials 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 5
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- 239000012588 trypsin Substances 0.000 description 5
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- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 description 3
- 239000001569 carbon dioxide Substances 0.000 description 3
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 3
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 3
- 229910000162 sodium phosphate Inorganic materials 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
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- 230000004927 fusion Effects 0.000 description 2
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
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- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 208000004680 Rectal Fistula Diseases 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 206010002156 anal fistula Diseases 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 210000003716 mesoderm Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
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- 238000010899 nucleation Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/10—Preservation of living parts
- A01N1/12—Chemical aspects of preservation
- A01N1/122—Preservation or perfusion media
- A01N1/125—Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
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- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
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Abstract
本发明公开了一种用于制备脂肪间充质干细胞的试剂盒，特征是盒内包括分别瓶装保存的脂肪保存液、脂肪洗涤液、脂肪分解液、细胞洗涤液、细胞培养液、细胞消化液、细胞冻存液。利用本发明试剂盒内的试剂，可以快速、安全获得大量脂肪间充质干细胞。The invention discloses a kit for preparing adipose mesenchymal stem cells, which is characterized in that the box includes a fat preservation solution, a fat washing solution, a lipolysis solution, a cell washing solution, a cell culture solution, and a cell digestion solution stored in bottles respectively. , Cell cryopreservation. Using the reagents in the kit of the present invention, a large amount of adipose mesenchymal stem cells can be quickly and safely obtained.
Description
Technical field
The present invention designs kit, more particularly to a kind of kit for being used to prepare fat mesenchymal stem cell.
Background technique
2001, there is a kind of cell with Multidirectional Differentiation potentiality in adipose tissue in the discovery such as Zuk, i.e., fills between fat
Matter stem cell, abbreviation fat stem cell.Its biological property is similar with mesenchymal stem cell, and can be to fat, bone, soft
The differentiation of the various kinds of cell such as bone, muscle, endothelium, hematopoiesis, liver, pancreas islet and nerve direction.
Adipose tissue rich reserves in human body, the easy wound of acquisition is small, in organizational project, organ reparation, gene therapy
Etc. suffer from wide application prospect, therefore fat mesenchymal stem cell has become the heat that stem cell field is concerned
Point.The autologous fat stem cell drug Cuepistem in January, 2012, South Korea FDA approval is successfully listed, for treating complexity
The concurrent anal fistula of clone disease.October in the same year, U.S. FDA have approved Cytori Therapeutics company fat stem cell to the heart
The clinical test of dirty failure patients' validity.Fat mesenchymal stem cell has wide potential applicability in clinical practice.Therefore, how simple
Single, quick, high-new, safety obtains significant quantities of fat mescenchymal stem cell as the task of top priority.
Summary of the invention
It is an object of the present invention to provide a kind of easy to operate, can quickly, safety obtain significant quantities of fat mescenchymal stem cell
Kit.
To achieve the above object, the present invention takes following design scheme: being used to prepare the kit of fat mesenchymal stem cell, box
Interior includes that the fat of bottled preservation respectively saves liquid, fatty cleaning solution, lipolysis liquid, cell washing solution, cell culture fluid, thin
Born of the same parents' digestive juice, cells frozen storing liquid.
It is containing 100 ~ 200ug/ml of penicillin, 100 ~ 200ug/ml of streptomysin, anphotericin 2.5 ~ 5 that the fat, which saves liquid,
The DMEM or DMEM/F12, MEM, RPMI- of μ g/ml, 1~2 μm of ol/ml of aspirin, 0.1~0.3nmol/ml of dexamethasone
1640 culture mediums.
The fat cleaning solution is containing 100 ~ 200ug/ml of penicillin, 100 ~ 200ug/ml of streptomysin, anphotericin 2.5 ~ 5
μ g/ml, 1~2 μm of ol/ml of aspirin, the physiological saline of 0.1~0.3nmol/ml of dexamethasone or PBS, D-Hanks,
HBSS。
The lipolysis liquid is the 0.75 ~ 2mg/ml of I containing clostridiopetidase A, 1~2 μm of ol/ml of aspirin, dexamethasone 0.1
The DMEM or DMEM/F12, MEM, RPMI-1640 culture medium of~0.3nmol/ml.
The cell washing solution be containing 1~2 μm of ol/ml of aspirin, 0.1~0.3nmol/ml of dexamethasone PBS or
D-Hanks、HBSS。
The cell culture fluid is the mesenchyma containing 1~2 μm of ol/ml of aspirin, 0.1~0.3nmol/ml of dexamethasone
Stem cell serum-free growth medium, the culture medium chemical component limit, and contain L-Glutamine, are free of phenol red and antibiotic.
The cell dissociation buffer be containing 1.25 ~ 2.5mg/ml of Trypsin-EDTA, containing 1~2 μm of ol/ml of aspirin,
The PBS or D-Hanks, HBSS solution of 0.1~0.3nmol/ml of Sai meter Song.
The cells frozen storing liquid is to contain 1~2 μm of ol/ml of aspirin, dexamethasone 0.1 containing 60 ~ 200mg/ml of DMSO
The mesenchymal stem cell serum-free growth medium of~0.3nmol/ml.
The present invention provides a kind of kit for preparing fat mesenchymal stem cell, in the kit, fat saves liquid
Effect is the activity guaranteed in the transportational process of fatty sample after collection, and is eliminated and pre- bacteriological protection, fungi.Fat washing
The effect of liquid is the blood cell that washing removes adipose tissue, while being eliminated and pre- bacteriological protection, fungi.The effect of lipolysis liquid
It is digestion adipose tissue, obtains single cell suspension.The effect of cell washing solution is the stem cell that washing obtains.Cell culture fluid is
Serum-free, and chemical component limits, and reduces the pathogenic infection risk of animal origin, culture fat mesenchymal stem cell relative to
Using safer for fetal calf serum.When the effect of cell dissociation buffer is cell passage, adherent mescenchymal stem cell is digested.Carefully
The effect of born of the same parents' frozen stock solution is the fat mesenchymal stem cell for freezing acquisition.
Using the kit, can obtaining significant quantities of fat mescenchymal stem cell in short period (in 3 weeks), (cell quantity is reachable
109The order of magnitude), and have easy to use, low in cost, the serum-free pathogenic infection risk of animal origin (reduce) is more pacified
Full feature will play a significant role in stem cell clinical test and its correlative study, and application prospect is extremely wide.
Specific embodiment
Method therefor is conventional method unless otherwise instructed in following embodiments, required reagent consumptive material and laboratory apparatus etc.
It can be bought by commercial sources.
Embodiment one is used to prepare the preparation and its separating effect detection of the kit of fat mesenchymal stem cell
One, preparation is used to prepare the kit of fat mesenchymal stem cell
1, solution allocation
The kit for being used to prepare fat mesenchymal stem cell of the invention includes following reagent
1) fat saves liquid configuration method: penicillin (brand Amresco, article No. 0242) 200mg, streptomysin (brand
Amresco, article No. 0382) 200mg, anphotericin (brand Amresco, article No. E437) 5mg, aspirin (Sigma,
A2093-100G) 0.1 μm of 2 mmol, dexamethasone (Sigma, D4902-25MG) ol, are dissolved in 1000ml DMEM culture medium
0.22um filtration sterilization.
2) fatty cleaning solution configuration method: penicillin 200mg, streptomysin 200mg, anphotericin 5mg, aspirin 2
0.1 μm of mmol, dexamethasone ol, are dissolved in 0.22um filtration sterilization in 1000ml physiological saline.
3) lipolysis liquid configuration method: clostridiopetidase A I(brand sigma, article No. C0130) 1 g, 2 mmol of aspirin,
0.1 μm of ol of dexamethasone is dissolved in 1000ml DMEM culture medium, 0.22um filter filtration sterilization.
4) cell washing solution configuration method: PBS is formulated as NaCl 8.50g, Na2HPO4·12H2O 3.58g,
NaH2PO4·2H2O 0.39g adds distilled water to 1000ml, high pressure sterilization.2 mmol of aspirin, dexamethasone 0.1
μm ol is dissolved in the above-mentioned PBS of 1000ml, 0.22um filter filtration sterilization.
5) cell culture fluid configuration method: serum-free basal medium (brand Lonza, article No. 00190620) 450ml
And its additive (brand Lonza, article No. 00192125) 50ml is mixed.0.1 μm of 2 mmol of aspirin, dexamethasone ol
It is dissolved in the serum free medium of 1000ml above method preparation, 0.22um filter filtration sterilization.
6) cell dissociation buffer configuration method: Trypsin(trypsase) 0.25g, EDTA-2Na 19.728mg is dissolved in above-mentioned
In the cell washing solution 100ml prepared, mix, 0.22um filter filtration sterilization.
7) cells frozen storing liquid configuration method: 10ml DMSO(brand sigma, article No. D2650) to be slowly added into 90ml above-mentioned
In the cell culture fluid prepared, mix gently.
1, the packing of each component
The specification of kit is 1 time/box, and the amount of each component is that fat saves 1 bottle of liquid (50ml/ bottles) in every box, fatty cleaning solution 1
Bottle (250ml/ bottles), 1 bottle of lipolysis liquid (50ml/ bottles), 3 bottles of cell washing solution (250ml/ bottles), 4 bottles of cell culture fluid
(250ml/ bottles), 1 bottle of cell dissociation buffer (50ml/ bottles), 1 bottle of cells frozen storing liquid (50ml/ bottles).
Each component in kit is dispensed according to mentioned reagent amount, is packaged to be that be used to prepare fat mesenchymal dry thin
The kit of born of the same parents.- 20 DEG C of kit preservations, validity period 2 years, 4 DEG C of refrigerators were interior before use or 37 DEG C of water-baths are thawed, and thawed it
4 DEG C of preservations afterwards are finished in one month.
Two, the preparation of fat mesenchymal stem cell
The kit of current step 1 prepares fat mesenchymal stem cell, and specific preparation method includes the following steps (with 20ml rouge
For fat, all maneuvering sequences are completed in clean bench):
1) fat transport: acquisition 20ml fat is added isometric fat and saves liquid, and 2 ~ 8 DEG C of constant temperature are stored in vaccine box,
It is sent in 48h to laboratory.
2) washing fat: inhaling abandoning fat and save liquid, and isometric fat cleaning solution is added, vibrates repeatedly several times, rouge after standing
Fat and fatty cleaning solution natural layering, inhale and abandon fatty cleaning solution, ibid, add isometric fatty cleaning solution oscillation washing 2 ~ 3
It is secondary, until underlying adipose cleaning solution is clarified, inhales and abandon fatty cleaning solution, obtain fat.
3) digestion fat: isometric lipolysis liquid (being preheating to 37 DEG C) is added in the fat of acquisition, is placed in constant temperature oscillation
In incubator, 37 DEG C, 200rpm, 30 ~ 60min is digested, until fat is in waste shape.
4) SVF(stromal vascular part is separated): shape adipose tissue is wasted by postdigestive, 700g is centrifuged 10min, uses pipette
Carefully remove from top to bottom upper layer grease and lower layer collagenase solution (pay attention to leaving above SVF precipitating a small amount of solution with
Exempt to disturb sedimentation cell), cell washing solution is added and mixes, it is 2.1 × 10 that 100um strainer filtering, which is drawn 1ml suspension and counted,7
A, 400g is centrifuged 8min, abandons supernatant, obtains SVF.
5) cell seeding: according to SVF cell number, according to 1.0 × 106Cell culture fluid 20ml is added in the density of a/ml, connects
Kind of 1 T175 culture bottle, is placed in carbon dioxide incubator, condition of culture: 37 ± 0.5 DEG C, carbon dioxide volume fraction is 5 ±
0.2%, it is changed the liquid once every 3d.
6) cell passes on: 7d or so, and primary cultured cell is inhaled up to when 70%~80% fusion and abandons old cell culture fluid, refinement
Born of the same parents' cleaning solution, stand 1min, inhale abandon cell washing solution, be added cell dissociation buffer, digestion time be 1.5~2.5min, then plus
Enter 2~3ml of cell culture fluid and stop digestion, blows and beats bottom of bottle repeatedly and largely fall off to cell, move into 50ml centrifuge tube, cell
It counts, is 4.6 × 107A, 400g is centrifuged 8min, abandons supernatant, obtains 4.6 × 107A P0 fat subsitutes mescenchymal stem cell.It presses
According to 5.0 × 105Cell culture fluid is added in/ml, is inoculated into 5 T175 culture bottles, is put into culture in carbon dioxide incubator, is calculated as
P1 generation.It, up to when 80%~90% fusion, ibid operates for cell to 3d or so, P1, harvests cell, count and obtain 1.9 × 108A P1
Fat subsitutes mescenchymal stem cell ibid continues passage (can pass within every 3 ~ 5 days primary), and so on, until harvest P3 generation, through thin
Born of the same parents are counted as 3.9 × 109A fat mesenchymal stem cell.
7) cell cryopreservation: by above-mentioned acquisition P3 for cell suspension, 400g is centrifuged 8min, abandons supernatant, is slowly added to cell jelly
Liquid storage mixes gently, and then in cryopreservation tube subscript clear-cells title, algebra and freezes the date；It is put into freezing storing box, -80 DEG C of refrigerators
Overnight, next day is transferred to liquid nitrogen and keeps for a long time, recovers when to be used.
As can be seen that within the short period (in 3 weeks), by only 20ml fat, being obtained a large amount of with kit of the invention
3.9 × 109A fat mesenchymal stem cell.Between showing that kit of the invention can obtain significant quantities of fat within a short period of time
Mesenchymal stem cells.
Embodiment two is used to prepare the preparation and its separating effect detection of the kit of fat mesenchymal stem cell
One, preparation is used to prepare the kit of fat mesenchymal stem cell
1, solution allocation
The kit for being used to prepare fat mesenchymal stem cell of the invention includes following reagent:
1) fat saves liquid configuration method: penicillin 100mg, streptomysin 100mg, anphotericin 2.5mg, aspirin 1
0.3 μm of mmol, dexamethasone ol, are dissolved in 1000ml DMEM/F12 culture medium.
2) fatty cleaning solution configuration method: penicillin 100mg, streptomysin 100mg, anphotericin 2.5mg, aspirin 1
0.3 μm of mmol, dexamethasone ol, are dissolved in 1000ml physiological saline.
3) lipolysis liquid configuration method: 0.3 μm of clostridiopetidase A I 2g, aspirin 1mmol, dexamethasone ol are dissolved in
In 1000ml DMEM/F12 culture medium, 0.22um filter filtration sterilization.
4) cell washing solution configuration method: PBS is formulated as NaCl 8.50g, Na2HPO4·12H2O3.58g, NaH2PO4·
2H2O 0.39g adds distilled water to 1000ml, high pressure sterilization.0.3 μm of 1 mmol of aspirin, dexamethasone ol are dissolved in
In above-mentioned configured PBS, 0.22um filter filtration sterilization.
5) cell culture fluid configuration method: serum-free basal medium (brand Lonza, article No. 00190620) 450ml
And its additive (brand Lonza, article No. 00192125) 50ml is mixed.1 mmol of aspirin, 0.3 μ of dexamethasone
Mol is dissolved in above-mentioned configured serum-free basal medium, 0.22um filter filtration sterilization.
6) cell dissociation buffer configuration method: Trypsin(trypsase) 125mg, EDTA-2Na 9.864mg is dissolved in above-mentioned
In the cell washing solution prepared, mix, 0.22um filter filtration sterilization.
7) cells frozen storing liquid configuration method: 10ml DMSO(brand sigma, article No. D2650) to be slowly added into 90ml above-mentioned
In the cell culture fluid prepared, mix gently.
2, the packing of each component
The specification of kit is 1 time/box, and the amount of each component is that fat saves 1 bottle of liquid (50ml/ bottles) in every box, fatty cleaning solution 1
Bottle (250ml/ bottles), 1 bottle of lipolysis liquid (50ml/ bottles), 3 bottles of cell washing solution (250ml/ bottles), 4 bottles of cell culture fluid
(250ml/ bottles), 1 bottle of cell dissociation buffer (50ml/ bottles), 1 bottle of cells frozen storing liquid (50ml/ bottles).
Each component in kit is dispensed according to mentioned reagent amount, is packaged to be that be used to prepare fat mesenchymal dry thin
The kit of born of the same parents.- 20 DEG C of kit preservations, validity period 2 years, 4 DEG C of refrigerators were interior before use or 37 DEG C of water-baths are thawed, and thawed it
4 DEG C of preservations afterwards are finished in one month.
Two, the preparation of fat mesenchymal stem cell
With the kit of step 1, method prepares fat mesenchymal stem cell in reference implementation example one, and with identical method pair
The mescenchymal stem cell of acquisition is counted, as a result 20ml fat, and amplification obtains 3.8 × 10 within 3 weeks9A fat mesenchymal is dry
Cell.Show that kit of the invention can obtain significant quantities of fat mescenchymal stem cell within a short period of time.
Embodiment three is used to prepare the preparation and its separating effect detection of the kit of fat mesenchymal stem cell
One, preparation is used to prepare the kit of fat mesenchymal stem cell
1, solution allocation
The kit for being used to prepare fat mesenchymal stem cell of the invention includes following reagent:
1) fat saves liquid configuration method: penicillin 150mg, streptomysin 150mg, anphotericin 2.5mg, aspirin 1.5
0.2 μm of mmol, dexamethasone ol, are dissolved in 1000ml DMEM culture medium.
2) fatty cleaning solution configuration method: penicillin 150mg, streptomysin 150mg, anphotericin 2.5mg, aspirin
0.2 μm of 1.5 mmol, dexamethasone ol, are dissolved in 1000ml physiological saline.
3) lipolysis liquid configuration method: 0.2 μm of 2 g of clostridiopetidase A I, 1.5 mmol of aspirin, dexamethasone ol are molten
In 1000ml DMEM culture medium, 0.22um filter filtration sterilization.
4) cell washing solution configuration method: PBS is formulated as NaCl 8.50g, Na2HPO4·12H2O 3.58g,
NaH2PO4·2H2O 0.39g adds distilled water to 1000ml, high pressure sterilization.1.5 mmol of aspirin, dexamethasone
0.2 μm of ol is dissolved in above-mentioned configured PBS, 0.22um filter filtration sterilization.
5) cell culture fluid configuration method: serum-free basal medium (brand Lonza, article No. 00190620) 450ml
And its additive (brand Lonza, article No. 00192125) 50ml is mixed.0.2 μm of 1.5 mmol of aspirin, dexamethasone ol
It is dissolved in above-mentioned configured serum-free basal medium, 0.22um filter filtration sterilization.
6) cell dissociation buffer configuration method: Trypsin(trypsase) 0.25g, EDTA-2Na 19.728mg is dissolved in above-mentioned
In the cell washing solution prepared, mix, 0.22um filter filtration sterilization.
7) cells frozen storing liquid configuration method: 15ml DMSO(brand sigma, article No. D2650) to be slowly added into 85ml above-mentioned
In the cell culture fluid prepared, mix gently.
3, the packing of each component
The specification of kit is 1 time/box, and the amount of each component is that fat saves 1 bottle of liquid (50ml/ bottles) in every box, fatty cleaning solution 1
Bottle (250ml/ bottles), 1 bottle of lipolysis liquid (50ml/ bottles), 3 bottles of cell washing solution (250ml/ bottles), 4 bottles of cell culture fluid
(250ml/ bottles), 1 bottle of cell dissociation buffer (50ml/ bottles), 1 bottle of cells frozen storing liquid (50ml/ bottles).
Each component in kit is dispensed according to mentioned reagent amount, is packaged to be that be used to prepare fat mesenchymal dry thin
The kit of born of the same parents.- 20 DEG C of kit preservations, validity period 2 years, 4 DEG C of refrigerators were interior before use or 37 DEG C of water-baths are thawed, and thawed it
4 DEG C of preservations afterwards are finished in one month.
Two, the preparation of fat mesenchymal stem cell
With the kit of step 1, method prepares fat mesenchymal stem cell in reference implementation example one, and with identical method pair
The mescenchymal stem cell of acquisition is counted, as a result 20ml fat, and amplification obtains 4.1 × 10 within 3 weeks9A fat mesenchymal is dry
Cell.Show that kit of the invention can obtain significant quantities of fat mescenchymal stem cell within a short period of time.
Example IV is used to prepare the preparation and its separating effect detection of the kit of fat mesenchymal stem cell
One, preparation is used to prepare the kit of fat mesenchymal stem cell
1, solution allocation
The kit for being used to prepare fat mesenchymal stem cell of the invention includes following reagent:
1) fat save liquid configuration method: penicillin 200mg, streptomysin 200mg, anphotericin 5mg, 1 mmol of aspirin,
0.1 μm of ol of dexamethasone, is dissolved in 1000ml DMEM culture medium.
2) fatty cleaning solution configuration method: penicillin 200mg, streptomysin 200mg, anphotericin 5mg, aspirin 1
0.1 μm of mmol, dexamethasone ol, are dissolved in 1000ml physiological saline.
3) lipolysis liquid configuration method: 0.1 μm of clostridiopetidase A I 1.5g, 1 mmol of aspirin, dexamethasone ol are dissolved in
In 1000ml DMEM culture medium, 0.22um filter filtration sterilization.
4) cell washing solution configuration method: 8.0 0.4 g of g, KCl of HBSS, formula NaCl, glucose 1 g, KH2PO4 60
mg、Na2HPO4 47.5 mg、NaHCO3 0.35g adds distilled water to 1000ml, adjusts PH between neutral 7.2-7.4, high pressure
Sterilizing.0.1 μm of 1 mmol of aspirin, dexamethasone ol are dissolved in 1000ml HBSS, 0.22um filter filtration sterilization
?.
5) cell culture fluid configuration method: serum-free basal medium (brand Lonza, article No. 00190620) 450ml
And its additive (brand Lonza, article No. 00192125) 50ml is mixed.1 mmol of aspirin, 0.1 μ of dexamethasone
Mol is dissolved in the above-mentioned configured serum-free basal medium of 1000ml, 0.22um filter filtration sterilization.
6) cell dissociation buffer configuration method: Trypsin(trypsase) 0.25g, EDTA-2Na 19.728mg is dissolved in above-mentioned
In the cell washing solution prepared, mix, 0.22um filter filtration sterilization.
7) cells frozen storing liquid configuration method: 10ml DMSO(brand sigma, article No. D2650) to be slowly added into 90ml above-mentioned
In the cell culture fluid prepared, mix gently.
4, the packing of each component
The specification of kit is 1 time/box, and the amount of each component is that fat saves 1 bottle of liquid (50ml/ bottles) in every box, fatty cleaning solution 1
Bottle (250ml/ bottles), 1 bottle of lipolysis liquid (50ml/ bottles), 3 bottles of cell washing solution (250ml/ bottles), 4 bottles of cell culture fluid
(250ml/ bottles), 1 bottle of cell dissociation buffer (50ml/ bottles), 1 bottle of cells frozen storing liquid (50ml/ bottles).
Each component in kit is dispensed according to mentioned reagent amount, is packaged to be that be used to prepare fat mesenchymal dry thin
The kit of born of the same parents.- 20 DEG C of kit preservations, validity period 2 years, 4 DEG C of refrigerators were interior before use or 37 DEG C of water-baths are thawed, and thawed it
4 DEG C of preservations afterwards are finished in one month.
Two, the preparation of fat mesenchymal stem cell
With the kit of step 1, method prepares fat mesenchymal stem cell in reference implementation example one, and with identical method pair
The mescenchymal stem cell of acquisition is counted, as a result 20ml fat, and amplification obtains 4.2 × 10 within 3 weeks9A fat mesenchymal is dry
Cell.Show that kit of the invention can obtain significant quantities of fat mescenchymal stem cell within a short period of time.
Embodiment five is used to prepare the preparation and its separating effect detection of the kit of fat mesenchymal stem cell
One, preparation is used to prepare the kit of fat mesenchymal stem cell
1, solution allocation
The kit for being used to prepare fat mesenchymal stem cell of the invention includes following reagent
1) fat save liquid configuration method: penicillin 200mg, streptomysin 200mg, anphotericin 5mg, 2 mmol of aspirin,
0.3 μm of ol of dexamethasone, is dissolved in 1000ml DMEM culture medium.
2) fatty cleaning solution configuration method: penicillin 200mg, streptomysin 200mg, anphotericin 5mg, aspirin 2
0.3 μm of mmol, dexamethasone ol, are dissolved in 1000ml physiological saline.
3) lipolysis liquid configuration method: 0.3 μm of 1.5 g of clostridiopetidase A I, 2 mmol of aspirin, dexamethasone ol are molten
In 1000ml DMEM culture medium, 0.22um filter filtration sterilization.
4) cell washing solution configuration method: D-Hanks is formulated KCl 0.40g, KH2PO40.06g, NaCl 8.00g,
NaHCO30.35g, Na2HPO4·12H2O 0.12g adds tri-distilled water to 1000ml, adjusts pH between 7.2-7.4, high pressure is gone out
Bacterium.0.3 μm of 2 mmol of aspirin, dexamethasone ol are dissolved in 1000ml D-Hanks, 0.22um filter filtration sterilization
?.
5) cell culture fluid configuration method: serum-free basal medium (brand Lonza, article No. 00190620) 450ml
And its additive (brand Lonza, article No. 00192125) 50ml is mixed.2 mmol of aspirin, 0.3 μ of dexamethasone
Mol is dissolved in above-mentioned configured serum-free basal medium, 0.22um filter filtration sterilization.
4) cell dissociation buffer configuration method: Trypsin(trypsase) 0.25g, EDTA-2Na 19.728mg is dissolved in above-mentioned
In the cell washing solution prepared, mix, 0.22um filter filtration sterilization.
5) cells frozen storing liquid configuration method: 10ml DMSO(brand sigma, article No. D2650) to be slowly added into 90ml above-mentioned
In the cell culture fluid prepared, mix gently.
2, the packing of each component
The specification of kit is 1 time/box, and the amount of each component is that fat saves 1 bottle of liquid (50ml/ bottles) in every box, fatty cleaning solution 1
Bottle (250ml/ bottles), 1 bottle of lipolysis liquid (50ml/ bottles), 3 bottles of cell washing solution (250ml/ bottles), 4 bottles of cell culture fluid
(250ml/ bottles), 1 bottle of cell dissociation buffer (50ml/ bottles), 1 bottle of cells frozen storing liquid (50ml/ bottles).
Each component in kit is dispensed according to mentioned reagent amount, is packaged to be that be used to prepare fat mesenchymal dry thin
The kit of born of the same parents.- 20 DEG C of kit preservations, validity period 2 years, 4 DEG C of refrigerators were interior before use or 37 DEG C of water-baths are thawed, and thawed it
4 DEG C of preservations afterwards are finished in one month.
Two, the preparation of fat mesenchymal stem cell
With the kit of step 1, method prepares fat mesenchymal stem cell in reference implementation example one, and with identical method pair
The mescenchymal stem cell of acquisition is counted, as a result 20ml fat, and amplification obtains 4.4 × 10 within 3 weeks9A fat mesenchymal is dry
Cell.Show that kit of the invention can obtain significant quantities of fat mescenchymal stem cell within a short period of time.
Claims (9)
1. being used to prepare the kit of fat mesenchymal stem cell, characterized in that include the fat guarantor of bottled preservation respectively in box
Liquid storage, fatty cleaning solution, lipolysis liquid, cell washing solution, cell culture fluid, cell dissociation buffer, cells frozen storing liquid.
2. the kit according to claim 1 for being used to prepare fat mesenchymal stem cell, characterized in that the kit
The amount of interior each component are as follows:
Fat saves 1 bottle of liquid, 50ml/ bottles；
1 bottle, 250ml/ bottles of fatty cleaning solution；
1 bottle of lipolysis liquid, 50ml/ bottles；
3 bottles of cell washing solution, 250ml/ bottles；
4 bottles of cell culture fluid, 250ml/ bottles；
1 bottle of cell dissociation buffer, 50ml/ bottles；
1 bottle of cells frozen storing liquid, 50ml/ bottles.
3. the kit according to claim 1 or 2 for being used to prepare fat mesenchymal stem cell, characterized in that the rouge
It is containing 100 ~ 200ug/ml of penicillin, 100 ~ 200ug/ml of streptomysin, 2.5 ~ 5 μ g/ml of anphotericin, aspirin that fat, which saves liquid,
The DMEM or DMEM/F12, MEM, RPMI-1640 culture medium of 1~2 μm of ol/ml, 0.1~0.3nmol/ml of dexamethasone.
4. the kit according to claim 1 or 2 for being used to prepare fat mesenchymal stem cell, characterized in that the rouge
Fat cleaning solution is containing 100 ~ 200ug/ml of penicillin, 100 ~ 200ug/ml of streptomysin, 2.5 ~ 5 μ g/ml of anphotericin, aspirin
The physiological saline or PBS, D-Hanks, HBSS of 1~2 μm of ol/ml, 0.1~0.3nmol/ml of dexamethasone.
5. the kit according to claim 1 or 2 for being used to prepare fat mesenchymal stem cell, characterized in that the rouge
Fat decomposed solution is the 0.75 ~ 2mg/ml of I containing clostridiopetidase A, aspirin 1~2 μm of ol/ml, 0.1~0.3nmol/ml of dexamethasone
DMEM or DMEM/F12, MEM, RPMI-1640 culture medium.
6. the kit according to claim 1 or 2 for being used to prepare fat mesenchymal stem cell, characterized in that described thin
Born of the same parents' cleaning solution is PBS or D-Hanks, HBSS containing 1~2 μm of ol/ml of aspirin, 0.1~0.3nmol/ml of dexamethasone.
7. the kit according to claim 1 or 2 for being used to prepare fat mesenchymal stem cell, characterized in that described thin
Born of the same parents' culture solution is that the mesenchymal stem cell serum-free containing 1~2 μm of ol/ml of aspirin, 0.1~0.3nmol/ml of dexamethasone is raw
Long culture medium, the culture medium chemical component limit, and contain L-Glutamine, are free of phenol red and antibiotic.
8. the kit according to claim 1 or 2 for being used to prepare fat mesenchymal stem cell, characterized in that described thin
Born of the same parents' digestive juice be containing 1.25 ~ 2.5mg/ml of Trypsin-EDTA, containing 1~2 μm of ol/ml of aspirin, dexamethasone 0.1~
The PBS or D-Hanks, HBSS solution of 0.3nmol/ml.
9. the kit according to claim 1 or 2 for being used to prepare fat mesenchymal stem cell, characterized in that described thin
Born of the same parents' frozen stock solution is containing 60 ~ 200mg/ml of DMSO, containing 1~2 μm of ol/ml of aspirin, 0.1~0.3nmol/ml of dexamethasone
Mesenchymal stem cell serum-free growth medium.
Priority Applications (1)
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| CN201710717884.6A CN109423476A (en) | 2017-08-21 | 2017-08-21 | It is used to prepare the kit of fat mesenchymal stem cell |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710717884.6A CN109423476A (en) | 2017-08-21 | 2017-08-21 | It is used to prepare the kit of fat mesenchymal stem cell |
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|---|---|
| CN109423476A true CN109423476A (en) | 2019-03-05 |
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Cited By (4)
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| CN109423473A (en) * | 2017-08-21 | 2019-03-05 | 青岛瑞思德生物科技有限公司 | It is used to prepare the kit of placenta stem-cell |
| CN109757469A (en) * | 2019-03-20 | 2019-05-17 | 江苏瑞思坦生物科技有限公司 | A kind of adipose tissue transport liquid and preparation method |
| CN109769799A (en) * | 2019-03-20 | 2019-05-21 | 江苏瑞思坦生物科技有限公司 | A kind of human fat tissue saves liquid and preparation method thereof |
| CN115927174A (en) * | 2022-12-25 | 2023-04-07 | 青岛瑞思德生物科技有限公司 | Efficient preparation kit for adipose-derived mesenchymal stem cells |
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