CN109423473A - It is used to prepare the kit of placenta stem-cell - Google Patents
It is used to prepare the kit of placenta stem-cell Download PDFInfo
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Abstract
The invention discloses a kind of kit for being used to prepare placenta stem-cell, kit includes that the placenta of bottled preservation respectively saves liquid, placenta cleaning solution I, placenta cleaning solution II, tissue breakdown liquid I, tissue breakdown liquid II, digestion terminate liquid, cell separating liquid, cells frozen storing liquid；A large amount of placenta stem-cells can be obtained within a short period of time using reagent in kit of the present invention, it is easy to use.
Description
Technical field
The invention belongs to stem cells technology fields, are related to a kind of kit for being used to prepare placenta stem-cell.
Background technique
Placenta stem-cell refers to the stem cell being present in placenta tissue.The placenta stem-cell for being currently known proof includes
A variety of stem cells such as candidate stem cell, mescenchymal stem cell, Subaerial blue green algae.In last decade, with placenta stem-cell industry
The continuous propulsion of appearance and the clinical application of change technology, in the world about the research achievement of placenta stem-cell present blowout it
Gesture.2008, M. Evangelista etc. delivered treatise and claims, and placenta stem-cell is this thing of the new hope of the following cell therapy
It is real.In subsequent several years, the research for placenta stem-cell occurs like the mushrooms after rain.2012, Auckland research institute, the U.S.
The high therapeutic application value that the researcher of children's hospital claims placenta stem-cell to have.2013, South Korea " CHA BIO&
DIOSTECH " stem-cell research professor's Wen Zhishu group issue a statement, claim for the first time confirm using result from human placenta at
Somatic stem cell can cure senile dementia.Celgene is the sixth-largest Biopharmaceutical Enterprises in the world, at present in medicament research and development
It is related to the drug of placenta stem-cell, mainly for Crohn disease, multiple sclerosis, arthritis, sarcoidosis, respectively one
Phase clinic and second phase are clinical.
Placental hematopoietic stem cell provides new source for hematopoietic stem cell transplantation.Candidate stem cell is red blood cell, white thin
The founder of born of the same parents, blood platelet etc. is the undifferentiated cell of height, is the initial cell of all haemocytes and immunocyte.Hematopoietic Stem
Cell transplantation can treat malignant hematologic disease, part malignant tumour, several fatal diseases of part genetic disease etc. ten.Quantity is rich
Richness is 5~10 times of Cord blood, breaches the bottleneck that Cord Blood-Derived candidate stem cell 30kg weight uses, and can be provided certainly
The enough quantity of somatic stem cell rebuilds the hemopoietic system of adult.
Placenta Subaerial blue green algae is derived from Subaerial blue green algae after the family embryo of newborn's placenta tissue, is sending out
It is close to educate stage and embryonic stem cell, has a histiocytic ability for being differentiated to form three germinal layers, but with embryonic stem cell
It is a difference in that and not will form teratoma, this characteristic is used directly for Subaerial blue green algae in vivo without worrying in body
Interior generation teratoma.Placenta Subaerial blue green algae has powerful proliferative capacity and multi-lineage potential.Be suitable for it is internal or
Have under vitro and be divided into mescenchymal stem cell, epithelial stem cell, neural stem cell, liver stem cells, myocyte, skeletonization are thin
The ability of the various kinds of cell such as born of the same parents, cartilage cell, stroma cell.It can be used to repair impaired or lesion histoorgan, the treatment heart,
A variety of diseases such as cranial vascular disease, the nervous system disease, liver diseases, bone tissue disease, corneal injury, empyrosis, myopathy.
Placenta mesenchyma stem cell not only has the general character i.e. unlimited multiplication capacity and multi-lineage potential of stem cell, also has
There is following advantage: 1) there is low immunogenicity and immunosuppressive action, this allows for the mescenchymal stem cell for the treatment of not only
Allosome can also be derived from from self；2) there is powerful immunoregulation effect, so that the mesenchyma of transplanting is dry thin
Born of the same parents can also adjust the immune state of patient while repairing damaged tissues；3) there is the spy to specific cells directed differentiation
Property, avoid the myeloid-lymphoid stem cells oncogenicity risk such as embryonic stem cell；4) source is very extensive, marrow, Placentas be rich in
Furthermore the position of mescenchymal stem cell can also be separated to MSC in fat, Cord blood and other adult tissue's organs.
Placenta stem-cell from start the just concern by countless experts in the world.Clinically carry out extensively at present and answers
With research, treatment-related disease includes: that aplastic anaemia, myelodysplastic syndrome, leukaemia etc. are pernicious
Or non-malignant hematologic disease, there are also type-II diabetes, idiopathic pulmonary fibrosis, ankylosing spondylitis, ulcerative colitis etc. are non-
Disease in the blood system.
In short, placenta stem-cell has wide potential applicability in clinical practice.Therefore, how simply, quickly, it is high-new, safety obtain
Obtaining a large amount of placenta stem-cells becomes the task of top priority.
Summary of the invention
It is an object of the present invention to provide one kind can be easy to operate, can obtain the examination of a large amount of placenta stem-cells quickly, safely
Agent box.
To achieve the above object, the technical solution adopted by the present invention is that: a kind of kit being used to prepare placenta stem-cell,
Placenta including bottled preservation respectively saves liquid, placenta cleaning solution I, placenta cleaning solution II, tissue breakdown liquid I, tissue breakdown liquid
II, terminate liquid, cell separating liquid, cells frozen storing liquid are digested.
It is containing 100~200ug/ml of penicillin, 100~200ug/ml of streptomysin, anphotericin 2.5 that the placenta, which saves liquid,
~5 μ g/ml, 0.5~1.5mg/ml of heparin sodium injection, 5~10mg/ml of human serum albumin, 1~2nmol/ml of aspirin,
0.1~0.3nmol/ml of dexamethasone, 2~3ng/ml of progesterone, 30~50nmol/ml of resveratrol, 10~30ug/ of insulin
DMEM, DMEM/F12, MEM or RPMI-1640 culture medium of ml, 2~4nmol/ml of vitamin C.
The placenta cleaning solution I is containing 100~200ug/ml of penicillin, 100~200ug/ml of streptomysin, anphotericin
2.5~5 μ g/ml, 0.5~1.5mg/ml of heparin sodium injection, 1~2nmol/ml of aspirin, dexamethasone 0.1~
0.3nmol/ml, 2~3ng/ml of progesterone, 30~50nmol/ml of resveratrol, 10~30ug/ml of insulin, vitamin C 2~
Physiological saline, PBS, D-Hanks or HBSS of 4nmol/ml.
The placenta cleaning solution II is containing 100~200ug/ml of penicillin, 100~200ug/ml of streptomysin, anphotericin
2.5~5 μ g/ml, 0.5~1.5mg/ml of heparin sodium injection, 5~10mg/ml of human serum albumin, 1~2nmol/ of aspirin
Ml, 0.1~0.3nmol/ml of dexamethasone, 2~3ng/ml of progesterone, 30~50nmol/ml of resveratrol, insulin 10~
30ug/ml, the physiological saline of 2~4nmol/ml of vitamin C, PBS, D-Hanks or HBSS.
The tissue breakdown liquid I is containing 0.5~2mg/ml of type Ⅳ collagenase, 1~2nmol/ml of aspirin, dexamethasone
0.1~0.3nmol/ml, 2~3ng/ml of progesterone, 30~50nmol/ml of resveratrol, 10~30ug/ml of insulin, vitamin C
DMEM, DMEM/F12, MEM or RPMI-1640 culture medium of 2~4nmol/ml.
The tissue breakdown liquid II be containing 0.5~2.5mg/ml of trypsase, 1~2nmol/ml of aspirin, fill in rice
0.1~0.3nmol/ml of pine, 2~3ng/ml of progesterone, 30~50nmol/ml of resveratrol, 10~30ug/ml of insulin, dimension life
DMEM, DMEM/F12, MEM or RPMI-1640 culture medium of plain 2~4nmol/ml of C.
The human serum albumin liquid that the digestion terminate liquid is concentration 0.2g/ml.
The cell separating liquid is the hydroxyethyl starch of depth 60mg/ml.
The cells frozen storing liquid is the KnockoutTM SR of 0.1~0.3mg/ml containing CryoSure-DEX40.
The present invention provides a kind of kits for preparing placenta stem-cell.In the kit, the effect that placenta saves liquid is to guarantee
Activity in the transportational process of placenta sample after collection, and eliminate and pre- bacteriological protection, fungi.Placenta cleaning solution I, placenta are washed
The effect for washing liquid II is the blood cell of washing removing placenta tissue, while eliminating and pre- bacteriological protection, fungi.Tissue breakdown liquid I,
The effect of tissue breakdown liquid II is digestion placenta tissue, obtains single cell suspension.The effect of digestion terminate liquid is to terminate tissue point
Solve the digestion of liquid.The effect of cell separating liquid removes red blood cell, obtains mononuclearcell.The effect of cells frozen storing liquid is to freeze
Deposit the placenta stem-cell of acquisition.
The kit for being used to prepare placenta stem-cell of the invention can obtain a large amount of placenta stem-cells in short 3h
(cell quantity is up to 109The order of magnitude), and have it is easy to use, low in cost, serum-free reduces the pathogen of animal origin
Infection risk, it is safer the features such as, will play a significant role in stem cell clinical test and its correlative study, application prospect pole
It is extensive.
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 use of the kit of placenta stem-cell
One, it is used to prepare the preparation of the kit of placenta stem-cell
1, solution allocation
The kit for being used to prepare placenta stem-cell of the invention includes following reagent:
(1) placenta saves liquid configuration method: penicillin (brand Amresco, article No. 0242) 100mg, streptomysin (brand
Amresco, article No. 0382) 100mg, anphotericin (brand Amresco, article No. E437) 2.5mg, human serum albumin (brand:
GRIFOLS, specification 20%:50ml) 2.5g, heparin sodium injection (producer: Chengdu Hai Tong pharmaceutcal corporation, Ltd, specification 2ml:
5000 units) 0.5g, 2 mmol of aspirin (Sigma, A2093-100G), 0.1 μ of dexamethasone (Sigma, D4902-25MG)
Mol, 2 μ g of progesterone (Sigma, V900699-5g), resveratrol (Sigma, R5010-100MG) 30 μm of ol, insulin
(Sigma, I0908) 10mg, vitamin C (Sigma, A5960-10mg) 2mmol are dissolved in 500ml RPMI-1640 culture medium
In.
(2) I configuration method of placenta cleaning solution: penicillin 200mg, streptomysin 200mg, anphotericin 5mg, heparin sodium injection
Liquid 1g, 2 mmol of aspirin, 0.1 μm of ol of dexamethasone, 2 μ g of progesterone, 30 μm of ol of resveratrol, insulin 10mg, vitamin
C 2mmol is dissolved in 1000ml physiological saline.
(3) II configuration method of placenta cleaning solution: penicillin 200mg, streptomysin 200mg, anphotericin 5mg, the white egg of people's blood
White 5g, heparin sodium injection 1g, 2 mmol of aspirin, 0.1 μm of ol of dexamethasone, 2 μ g of progesterone, resveratrol 30 μm of ol, pancreases
Island element 10mg, vitamin C 2mmol, are dissolved in 1000ml physiological saline.
(4) I configuration method of tissue breakdown liquid: type Ⅳ collagenase (brand sigma, article No. C5138) 1 g, aspirin 2
Mmol, 0.1 μm of ol of dexamethasone, 2 μ g of progesterone, 30 μm of ol of resveratrol, insulin 10mg, vitamin C 2mmol are dissolved in
In 500ml RPMI-1640 culture medium, 0.22um filter filtration sterilization.
(5) II configuration method of tissue breakdown liquid: trypsase 1.25g, 2 mmol of aspirin, 0.1 μm of ol of dexamethasone,
2 μ g of progesterone, 30 μm of ol of resveratrol, insulin 10mg, vitamin C 2mmol are dissolved in 500ml RPMI-1640 culture medium,
0.22um filter filtration sterilization.
(6) digest terminate liquid configuration method: the commercialization concentration 0.2g/ml of purchase human serum albumin (brand:
GRIFOLS, specification 20%:50ml).
(7) cell separating liquid configuration method: the commercialization 60mg/ml hydroxyethyl starch (producer: Beijing Fresenius of purchase
Card is than Pharmaceuticals Ltd, specification 500ml:30g hydroxyethyl starch 130/0.4 and 4.5g sodium chloride).
(8) cells frozen storing liquid configuration method: 20ml CryoSure-DEX40(brand WAK, specification 50ml) it is slowly added into
80ml KnockoutTMSR(brand Life Technologies, article No. N10828028) in, it mixes gently.
2, the packing of each component
The specification of kit is 1 time/box, the amount of each component in every box are as follows: placenta saves 1 bottle of liquid (500ml/ bottles), placenta washing
I 2 bottles of liquid (500ml/ bottles), II 2 bottles of placenta cleaning solution (500ml/ bottles), I 1 bottles of tissue breakdown liquid (500ml/ bottles), tissue breakdown
II 1 bottles of liquid (300ml/ bottles) digests 1 bottle of terminate liquid (20ml/ bottles), 1 bottle of cell separating liquid (100ml/ bottles), cells frozen storing liquid 1
Bottle (50ml/ bottles).
Each component in kit is dispensed according to mentioned reagent amount, is packaged to be the examination for being used to prepare placenta stem-cell
Agent box.Placenta saves liquid, placenta cleaning solution I, placenta cleaning solution II, tissue breakdown liquid I, tissue breakdown liquid II, thin in kit
Born of the same parents' frozen stock solution is saved in -20 DEG C, and validity period 2 years, 4 DEG C of refrigerators were interior before use or 37 DEG C of water-baths are thawed, 4 DEG C of guarantors after defrosting
It deposits, is finished in one month；Digestion terminate liquid, cell separating liquid are kept in dark place in room temperature in kit, and validity period is 2 years.
Two, the application of standby placenta stem-cell kit
Current kit manufactured in the present embodiment prepares placenta stem-cell, and specific preparation method includes the following steps, with a complete
For whole placenta, all operations step is completed in clean bench:
1, placenta transports: a complete placenta of acquisition is added suitable placenta and saves liquid, is advisable with submerging placenta, and 2~8
DEG C constant temperature is stored in vaccine box, for 24 hours in send to laboratory.
2, it washs placenta: inhaling and abandon placenta preservation liquid, the placenta cleaning solution I of appropriate volume is added, vibrate repeatedly several times, inhale and abandon
Placenta cleaning solution I ibid adds the oscillation of placenta cleaning solution I washing 2~3 times of appropriate volume, until the bloodstain on placenta surface
Washes clean inhales and abandons placenta cleaning solution I；Amnion and the decidua for removing placenta surface, are added the placenta cleaning solution II of appropriate volume,
Placenta tissue is shredded, and the blood in tissue block is cleaned up, collects placenta cleaning solution II, is centrifuged in 1800rpm room temperature
10min abandons supernatant, cell precipitation is resuspended with 50ml placenta cleaning solution II, by placenta cleaning solution II: cell separating liquid=4:1 ratio
Cell separating liquid is added in example, is layered after being stored at room temperature 30min after mixing, collects supernatant liquid.
3, it digests placenta: collecting the placenta tissue block shredded, addition and the isometric tissue breakdown liquid I of placenta tissue block,
20~30min is digested in 37 DEG C of shaking baths, the tissue breakdown liquid II of 1/4 volume of placenta tissue block is added later, in 37
10~20min is digested in DEG C shaking bath.Filtrate is collected by filtration through 200 mesh analysis sieve in placenta tissue block after digesting twice, adds
Enter to digest terminate liquid 10ml, be centrifuged 10min in 1800rpm room temperature after mixing, abandon supernatant, is resuspended with suitable placenta cleaning solution II
Cell precipitation, in placenta cleaning solution II: cell separating liquid is added in cell separating liquid=4:1 ratio, after mixing in 300rpm
Room temperature is centrifuged 10min, collects supernatant liquid.
4, it harvests cell: merging the supernatant liquid collected in step 2 and 3, mononuclearcell is measured in blood analyser
Quantity be 4.06 × 109It is a.
5, cell cryopreservation: above-mentioned acquisition cell suspension is centrifuged 10min in 1800rpm room temperature, abandons supernatant, is slowly added to thin
Born of the same parents' frozen stock solution, mixes gently, and then shows the essential information of cell on freezing bag, after being down to -80 DEG C in Programmed freezing instrument,
It is transferred in liquid nitrogen and saves for a long time.It recovers when to be used.
As can be seen that in short period 3h, by a complete placenta, can get a large amount of with kit of the invention
Placenta stem-cell, show that kit of the invention can obtain a large amount of placenta stem-cells within a short period of time.
Embodiment two is used to prepare the preparation and application of the kit of placenta stem-cell
One, it is used to prepare the preparation of the kit of placenta stem-cell
1, solution allocation
The kit for being used to prepare placenta stem-cell of the invention includes following reagent:
(1) placenta saves liquid configuration method: penicillin 50mg, streptomysin 50mg, anphotericin 1.25mg, human serum albumin
1.25g, heparin sodium injection 0.25g, aspirin 2mmol, 0.3 μm of ol of dexamethasone, 3 μ g of progesterone, 50 μm of ol of resveratrol,
Insulin 3 0mg, vitamin C 4mmol are dissolved in 500ml DMEM culture medium.
(2) I configuration method of placenta cleaning solution: penicillin 100mg, streptomysin 100mg, anphotericin 2.5mg, heparin sodium note
Penetrate liquid 0.5g, aspirin 2mmol, 0.3 μm of ol of dexamethasone, 3 μ g of progesterone, 50 μm of ol of resveratrol, Insulin 3 0mg, dimension life
Plain C 4mmol, is dissolved in 1000mlPBS.
(3) II configuration method of placenta cleaning solution: penicillin 100mg, streptomysin 100mg, anphotericin 2.5mg, people's blood are white
Albumen 2.5g, heparin sodium injection 0.5g, aspirin 2mmol, 0.3 μm of ol of dexamethasone, 3 μ g of progesterone, 50 μ of resveratrol
Mol, Insulin 3 0mg, vitamin C 4mmol, are dissolved in 1000mlPBS.
(4) I configuration method of tissue breakdown liquid: 1 g of type Ⅳ collagenase, aspirin 2mmol, 0.3 μm of ol of dexamethasone, pregnant
3 μ g of ketone, 50 μm of ol of resveratrol, Insulin 3 0mg, vitamin C 4mmol are dissolved in 500ml DMEM culture medium, 0.22um filter
Device filtration sterilization.
(5) II configuration method of tissue breakdown liquid: trypsase 1.25g, aspirin 2mmol, 0.3 μm of ol of dexamethasone,
3 μ g of progesterone, 50 μm of ol of resveratrol, Insulin 3 0mg, vitamin C 4mmol are dissolved in 500ml DMEM culture medium, 0.22um
Filter filtration sterilization.
(6) digest terminate liquid configuration method: the commercialization concentration 0.2g/ml of purchase human serum albumin (brand:
GRIFOLS, specification 20%:50ml).
(7) cell separating liquid configuration method: the commercialization 60mg/ml hydroxyethyl starch (producer: Beijing Fresenius of purchase
Card is than Pharmaceuticals Ltd, specification 500ml:30g hydroxyethyl starch 130/0.4 and 4.5g sodium chloride).
(8) cells frozen storing liquid configuration method: 20ml CryoSure-DEX40 is slowly added into 80ml KnockoutTM SR
In, it mixes gently.
2, the packing of each component
The specification of kit is 1 time/box, the amount of each component in every box are as follows: placenta saves 1 bottle of liquid (500ml/ bottles), placenta washing
I 2 bottles of liquid (500ml/ bottles), II 2 bottles of placenta cleaning solution (500ml/ bottles), I 1 bottles of tissue breakdown liquid (500ml/ bottles), tissue breakdown
II 1 bottles of liquid (300ml/ bottles) digests 1 bottle of terminate liquid (20ml/ bottles), 1 bottle of cell separating liquid (100ml/ bottles), cells frozen storing liquid 1
Bottle (50ml/ bottles).
Each component in kit is dispensed according to mentioned reagent amount, is packaged to be the examination for being used to prepare placenta stem-cell
Agent box.Placenta saves liquid, placenta cleaning solution I, placenta cleaning solution II, tissue breakdown liquid I, tissue breakdown liquid II, thin in kit
Born of the same parents' frozen stock solution is saved in -20 DEG C, and validity period 2 years, 4 DEG C of refrigerators were interior before use or 37 DEG C of water-baths are thawed, 4 DEG C of guarantors after defrosting
It deposits, is finished in one month；Digestion terminate liquid, cell separating liquid are kept in dark place in room temperature in kit, and validity period is 2 years.
Two, the application of standby placenta stem-cell kit
Kit manufactured in the present embodiment, method prepares placenta stem-cell in reference implementation example one, and with identical method to obtaining
The placenta stem-cell obtained is counted, and as a result a complete placenta can be obtained 4.18 × 10 by the separation of 3h9It is a single
Nucleus shows that kit of the invention can obtain a large amount of placenta stem-cells within a short period of time.
Embodiment three is used to prepare the preparation and application of the kit of placenta stem-cell
One, it is used to prepare the preparation of the kit of placenta stem-cell
1, solution allocation
The kit for being used to prepare placenta stem-cell of the invention includes following reagent:
(1) placenta saves liquid configuration method: penicillin 100mg, streptomysin 100mg, anphotericin 2.5mg, human serum albumin
2.5g, heparin sodium injection 0.5g, aspirin 2mmol, 0.1 μm of ol of dexamethasone, 2 μ g of progesterone, resveratrol 40 μm of ol, pancreases
Island element 20mg, vitamin C 3mmol are dissolved in 500ml RPMI-1640 culture medium.
(2) I configuration method of placenta cleaning solution: penicillin 200mg, streptomysin 200mg, anphotericin 5mg, heparin sodium injection
Liquid 1g, aspirin 2mmol, 0.1 μm of ol of dexamethasone, 2 μ g of progesterone, 40 μm of ol of resveratrol, insulin 20mg, vitamin C
3mmol is dissolved in 1000mlPBS.
(3) II configuration method of placenta cleaning solution: penicillin 200mg, streptomysin 200mg, anphotericin 5mg, the white egg of people's blood
White 5g, heparin sodium injection 1g, aspirin 2mmol, 0.1 μm of ol of dexamethasone, 2 μ g of progesterone, resveratrol 40 μm of ol, pancreas islet
Plain 20mg, vitamin C 3mmol, are dissolved in 1000mlPBS.
(4) I configuration method of tissue breakdown liquid: type Ⅳ collagenase 0.5g, aspirin 2mmol, 0.1 μm of ol of dexamethasone,
2 μ g of progesterone, 40 μm of ol of resveratrol, insulin 20mg, vitamin C 3mmol are dissolved in 500ml RPMI-1640 culture medium,
0.22um filter filtration sterilization.
(5) II configuration method of tissue breakdown liquid: trypsase 1.25g, aspirin 2mmol, 0.1 μm of ol of dexamethasone,
2 μ g of progesterone, 40 μm of ol of resveratrol, insulin 20mg, vitamin C 3mmol are dissolved in 500ml RPMI-1640 culture medium,
0.22um filter filtration sterilization.
(6) digest terminate liquid configuration method: the commercialization concentration 0.2g/ml of purchase human serum albumin (brand:
GRIFOLS, specification 20%:50ml).
(7) cell separating liquid configuration method: the commercialization 60mg/ml hydroxyethyl starch (producer: Beijing Fresenius of purchase
Card is than Pharmaceuticals Ltd, specification 500ml:30g hydroxyethyl starch 130/0.4 and 4.5g sodium chloride).
(8) cells frozen storing liquid configuration method: 20ml CryoSure-DEX40 is slowly added into 80ml KnockoutTM SR
In, it mixes gently.
2, the packing of each component
The specification of kit is 1 time/box, the amount of each component in every box are as follows: placenta saves 1 bottle of liquid (500ml/ bottles), placenta washing
I 2 bottles of liquid (500ml/ bottles), II 2 bottles of placenta cleaning solution (500ml/ bottles), I 1 bottles of tissue breakdown liquid (500ml/ bottles), tissue breakdown
II 1 bottles of liquid (300ml/ bottles) digests 1 bottle of terminate liquid (20ml/ bottles), 1 bottle of cell separating liquid (100ml/ bottles), cells frozen storing liquid 1
Bottle (50ml/ bottles).
Each component in kit is dispensed according to mentioned reagent amount, is packaged to be the examination for being used to prepare placenta stem-cell
Agent box.Placenta saves liquid, placenta cleaning solution I, placenta cleaning solution II, tissue breakdown liquid I, tissue breakdown liquid II, thin in kit
Born of the same parents' frozen stock solution is saved in -20 DEG C, and validity period 2 years, 4 DEG C of refrigerators were interior before use or 37 DEG C of water-baths are thawed, 4 DEG C of guarantors after defrosting
It deposits, is finished in one month；Digestion terminate liquid, cell separating liquid are kept in dark place in room temperature in kit, and validity period is 2 years.
Two, the application of standby placenta stem-cell kit
With the kit of step 1, method prepares placenta stem-cell in reference implementation example one, and with identical method to acquisition
Placenta stem-cell is counted, and as a result a complete placenta can be obtained 3.83 × 10 by the separation of 3h9A single core is thin
Born of the same parents show that kit of the invention can obtain a large amount of placenta stem-cells within a short period of time.
Example IV is used to prepare the preparation and application of the kit of placenta stem-cell
One, it is used to prepare the preparation of the kit of placenta stem-cell
1, solution allocation
The kit for being used to prepare placenta stem-cell of the invention includes following reagent:
(1) placenta saves liquid configuration method: penicillin 100mg, streptomysin 100mg, anphotericin 2.5mg, human serum albumin
2.5g, heparin sodium injection 0.5g, aspirin 1.5mmol, 0.1 μm of ol of dexamethasone, 2.5 μ g of progesterone, 35 μ of resveratrol
Mol, insulin 12mg, vitamin C 2.6mmol are dissolved in 500ml DMEM/F12 culture medium.
(2) I configuration method of placenta cleaning solution: penicillin 200mg, streptomysin 200mg, anphotericin 5mg, heparin sodium injection
Liquid 1g, aspirin 1.5mmol, 0.1 μm of ol of dexamethasone, 2.5 μ g of progesterone, 35 μm of ol of resveratrol, insulin 12mg, dimension life
Plain C 2.6mmol, is dissolved in 1000mlPBS.
(3) II configuration method of placenta cleaning solution: penicillin 200mg, streptomysin 200mg, anphotericin 5mg, the white egg of people's blood
White 5g, heparin sodium injection 1g, aspirin 1.5mmol, 0.1 μm of ol of dexamethasone, 2.5 μ g of progesterone, 35 μm of ol of resveratrol,
Insulin 12mg, vitamin C 2.6mmol, are dissolved in 1000mlPBS.
(4) I configuration method of tissue breakdown liquid: type Ⅳ collagenase 0.25g, aspirin 1.5mmol, 0.1 μ of dexamethasone
Mol, 2.5 μ g of progesterone, 35 μm of ol of resveratrol, insulin 12mg, vitamin C 2.6mmol are dissolved in 500ml DMEM/F12 culture
In base, 0.22um filter filtration sterilization.
(5) II configuration method of tissue breakdown liquid: trypsase 1.25g, aspirin 1.5mmol, 0.1 μ of dexamethasone
Mol, 2.5 μ g of progesterone, 35 μm of ol of resveratrol, insulin 12mg, vitamin C 2.6mmol are dissolved in 500ml DMEM/F12 culture
In base, 0.22um filter filtration sterilization.
(6) digest terminate liquid configuration method: the commercialization concentration 0.2g/ml of purchase human serum albumin (brand:
GRIFOLS, specification 20%:50ml).
(7) cell separating liquid configuration method: the commercialization 60mg/ml hydroxyethyl starch (producer: Beijing Fresenius of purchase
Card is than Pharmaceuticals Ltd, specification 500ml:30g hydroxyethyl starch 130/0.4 and 4.5g sodium chloride).
(8) cells frozen storing liquid configuration method: 20ml CryoSure-DEX40 is slowly added into 80ml KnockoutTM SR
In, it mixes gently.
2, the packing of each component
The specification of kit is 1 time/box, the amount of each component in every box are as follows: placenta saves 1 bottle of liquid (500ml/ bottles), placenta washing
I 2 bottles of liquid (500ml/ bottles), II 2 bottles of placenta cleaning solution (500ml/ bottles), I 1 bottles of tissue breakdown liquid (500ml/ bottles), tissue breakdown
II 1 bottles of liquid (300ml/ bottles) digests 1 bottle of terminate liquid (20ml/ bottles), 1 bottle of cell separating liquid (100ml/ bottles), cells frozen storing liquid 1
Bottle (50ml/ bottles).
Each component in kit is dispensed according to mentioned reagent amount, is packaged to be the examination for being used to prepare placenta stem-cell
Agent box.Placenta saves liquid, placenta cleaning solution I, placenta cleaning solution II, tissue breakdown liquid I, tissue breakdown liquid II, thin in kit
Born of the same parents' frozen stock solution is saved in -20 DEG C, and validity period 2 years, 4 DEG C of refrigerators were interior before use or 37 DEG C of water-baths are thawed, 4 DEG C of guarantors after defrosting
It deposits, is finished in one month；Digestion terminate liquid, cell separating liquid are kept in dark place in room temperature in kit, and validity period is 2 years.
Two, the application of standby placenta stem-cell kit
With kit manufactured in the present embodiment, method prepares placenta stem-cell in reference implementation example one, and with identical method pair
The placenta stem-cell of acquisition is counted, and as a result a complete placenta can be obtained 3.91 × 10 by the separation of 3h9A list
A nucleus shows that kit of the invention can obtain a large amount of placenta stem-cells within a short period of time.
Embodiment five is used to prepare the preparation and application of the kit of placenta stem-cell
One, it is used to prepare the preparation of the kit of placenta stem-cell
1, solution allocation
The kit for being used to prepare placenta stem-cell of the invention includes following reagent:
(1) placenta saves liquid configuration method: penicillin 100mg, streptomysin 100mg, anphotericin 2.5mg, human serum albumin
2.5g, heparin sodium injection 0.5g, aspirin 2mmol, 0.3 μm of ol of dexamethasone, 3 μ g of progesterone, resveratrol 50 μm of ol, pancreases
Island element 30mg, vitamin C 4mmol are dissolved in 500ml RPMI-1640 culture medium.
(2) I configuration method of placenta cleaning solution: penicillin 200mg, streptomysin 200mg, anphotericin 5mg, heparin sodium injection
Liquid 1g, aspirin 2mmol, 0.3 μm of ol of dexamethasone, 3 μ g of progesterone, 50 μm of ol of resveratrol, Insulin 3 0mg, vitamin C
4mmol is dissolved in 1000mlPBS.
(3) II configuration method of placenta cleaning solution: penicillin 200mg, streptomysin 200mg, anphotericin 5mg, the white egg of people's blood
White 5g, heparin sodium injection 1g, aspirin 2mmol, 0.3 μm of ol of dexamethasone, 3 μ g of progesterone, resveratrol 50 μm of ol, pancreas islet
Plain 30mg, vitamin C 4mmol, are dissolved in 1000mlPBS.
(4) I configuration method of tissue breakdown liquid: type Ⅳ collagenase 1g, aspirin 2mmol, 0.3 μm of ol of dexamethasone, pregnant
3 μ g of ketone, 50 μm of ol of resveratrol, Insulin 3 0mg, vitamin C 4mmol are dissolved in 500ml RPMI-1640 culture medium,
0.22um filter filtration sterilization.
(5) II configuration method of tissue breakdown liquid: trypsase 0.25g, aspirin 2mmol, 0.3 μm of ol of dexamethasone,
3 μ g of progesterone, 50 μm of ol of resveratrol, Insulin 3 0mg, vitamin C 4mmol are dissolved in 500ml RPMI-1640 culture medium,
0.22um filter filtration sterilization.
(6) digest terminate liquid configuration method: the commercialization concentration 0.2g/ml of purchase human serum albumin (brand:
GRIFOLS, specification 20%:50ml).
(7) cell separating liquid configuration method: the commercialization 60mg/ml hydroxyethyl starch (producer: Beijing Fresenius of purchase
Card is than Pharmaceuticals Ltd, specification 500ml:30g hydroxyethyl starch 130/0.4 and 4.5g sodium chloride).
(8) cells frozen storing liquid configuration method: 20ml CryoSure-DEX40 is slowly added into 80ml KnockoutTM SR
In, it mixes gently.
2, the packing of each component
The specification of kit is 1 time/box, the amount of each component in every box are as follows: placenta saves 1 bottle of liquid (500ml/ bottles), placenta washing
I 2 bottles of liquid (500ml/ bottles), II 2 bottles of placenta cleaning solution (500ml/ bottles), I 1 bottles of tissue breakdown liquid (500ml/ bottles), tissue breakdown
II 1 bottles of liquid (300ml/ bottles) digests 1 bottle of terminate liquid (20ml/ bottles), 1 bottle of cell separating liquid (100ml/ bottles), cells frozen storing liquid 1
Bottle (50ml/ bottles).
Each component in kit is dispensed according to mentioned reagent amount, is packaged to be the examination for being used to prepare placenta stem-cell
Agent box.Placenta saves liquid, placenta cleaning solution I, placenta cleaning solution II, tissue breakdown liquid I, tissue breakdown liquid II, thin in kit
Born of the same parents' frozen stock solution is saved in -20 DEG C, and validity period 2 years, 4 DEG C of refrigerators were interior before use or 37 DEG C of water-baths are thawed, 4 DEG C of guarantors after defrosting
It deposits, is finished in one month；Digestion terminate liquid, cell separating liquid are kept in dark place in room temperature in kit, and validity period is 2 years.
Two, the application of standby placenta stem-cell kit
With kit manufactured in the present embodiment, method prepares placenta stem-cell in reference implementation example one, and with identical method pair
The placenta stem-cell of acquisition is counted, and as a result a complete placenta can be obtained 3.77 × 10 by the separation of 3h9A list
A nucleus shows that kit of the invention can obtain a large amount of placenta stem-cells within a short period of time.
Claims (10)
1. the kit of placenta stem-cell is used to prepare, it is characterized in that: including placenta the preservation liquid, tire of bottled preservation respectively in box
Disk cleaning solution I, placenta cleaning solution II, tissue breakdown liquid I, tissue breakdown liquid II, digestion terminate liquid, cell separating liquid, cell freeze
Liquid storage.
2. the kit according to claim 1 for being used to prepare placenta stem-cell, characterized in that each group in the kit
The amount divided are as follows:
Placenta saves 1 bottle of liquid, 500ml/ bottles；
I 2 bottles of placenta cleaning solution, 500ml/ bottles；
II 2 bottles of placenta cleaning solution, 500ml/ bottles；
I 1 bottles of tissue breakdown liquid, 500ml/ bottles；
II 1 bottles of tissue breakdown liquid, 300ml/ bottles；
1 bottle of terminate liquid, 20ml/ bottles of digestion；
1 bottle of cell separating liquid, 100ml/ bottles；
1 bottle of cells frozen storing liquid, 50ml/ bottles.
3. the kit according to claim 1 or 2 for being used to prepare placenta stem-cell, it is characterized in that: the placenta saves
Liquid is containing 100~200ug/ml of penicillin, 100~200ug/ml of streptomysin, 2.5~5 μ g/ml of anphotericin, heparin sodium injection
0.5~1.5mg/ml of liquid, 5~10mg/ml of human serum albumin, 1~2nmol/ml of aspirin, dexamethasone 0.1~
0.3nmol/ml, 2~3ng/ml of progesterone, 30~50nmol/ml of resveratrol, 10~30ug/ml of insulin, vitamin C 2~
DMEM, DMEM/F12, MEM or RPMI-1640 culture medium of 4nmol/ml.
4. the kit according to claim 1 or 2 for being used to prepare placenta stem-cell, it is characterized in that: the placenta washs
Liquid I is containing 100~200ug/ml of penicillin, 100~200ug/ml of streptomysin, 2.5~5 μ g/ml of anphotericin, heparin sodium injection
It is 0.5~1.5mg/ml of liquid, 1~2nmol/ml of aspirin, 0.1~0.3nmol/ml of dexamethasone, 2~3ng/ml of progesterone, white
30~50nmol/ml of veratryl alcohol, 10~30ug/ml of insulin, the physiological saline of 2~4nmol/ml of vitamin C, PBS, D-
Hanks or HBSS.
5. the kit according to claim 1 or 2 for being used to prepare placenta stem-cell, it is characterized in that: the placenta washs
Liquid II is containing 100~200ug/ml of penicillin, 100~200ug/ml of streptomysin, 2.5~5 μ g/ml of anphotericin, heparin sodium note
Penetrate 0.5~1.5mg/ml of liquid, 5~10mg/ml of human serum albumin, 1~2nmol/ml of aspirin, dexamethasone 0.1~
0.3nmol/ml, 2~3ng/ml of progesterone, 30~50nmol/ml of resveratrol, 10~30ug/ml of insulin, vitamin C 2~
Physiological saline, PBS, D-Hanks or HBSS of 4nmol/ml.
6. the kit according to claim 1 or 2 for being used to prepare placenta stem-cell, it is characterized in that: the tissue breakdown
Liquid I is containing 0.5~2mg/ml of type Ⅳ collagenase, 1~2nmol/ml of aspirin, 0.1~0.3nmol/ml of dexamethasone, progesterone
2~3ng/ml, 30~50nmol/ml of resveratrol, 10~30ug/ml of insulin, 2~4nmol/ml of vitamin C DMEM,
DMEM/F12, MEM or RPMI-1640 culture medium.
7. the kit according to claim 1 or 2 for being used to prepare placenta stem-cell, it is characterized in that: the tissue breakdown
Liquid II is containing 0.5~2.5mg/ml of trypsase, 1~2nmol/ml of aspirin, 0.1~0.3nmol/ml of dexamethasone, pregnant
2~3ng/ml of ketone, 30~50nmol/ml of resveratrol, 10~30ug/ml of insulin, vitamin C 2~4nmol/ml
DMEM, DMEM/F12, MEM or RPMI-1640 culture medium.
8. the kit according to claim 1 or 2 for being used to prepare placenta stem-cell, it is characterized in that: the digestion terminates
Liquid is the human serum albumin liquid of concentration 0.2g/ml.
9. the kit according to claim 1 or 2 for being used to prepare placenta stem-cell, it is characterized in that: the cell separates
Liquid is the hydroxyethyl starch of concentration 60mg/ml.
10. the kit according to claim 1 or 2 for being used to prepare placenta stem-cell, it is characterized in that: the cell cryopreservation
Liquid is the Knockout of 0.1~0.3mg/ml containing CryoSure-DEX40TM SR。
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