CN109423477A - It is used to prepare the kit of umbilical cord mesenchymal stem cells - Google Patents
It is used to prepare the kit of umbilical cord mesenchymal stem cells Download PDFInfo
- Publication number
- CN109423477A CN109423477A CN201710718154.8A CN201710718154A CN109423477A CN 109423477 A CN109423477 A CN 109423477A CN 201710718154 A CN201710718154 A CN 201710718154A CN 109423477 A CN109423477 A CN 109423477A
- Authority
- CN
- China
- Prior art keywords
- umbilical cord
- mesenchymal stem
- bottles
- stem cells
- cell
- 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.)
- Pending
Links
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
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
-
- 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
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/33—Insulin
-
- 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
- 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
- C12N2501/392—Sexual steroids
Abstract
The invention discloses a kind of kits for being used to prepare umbilical cord mesenchymal stem cells, it is characterized in umbilical cord preserving fluid, umbilical cord cleaning solution, umbilical cord decomposed solution, cell washing solution, cell culture fluid, the cell dissociation buffer, cells frozen storing liquid including bottled preservation respectively in box, a large amount of umbilical cord mesenchymal stem cells can be obtained in the short period using the reagent in kit of the present invention, it is esy to use, safe and reliable.
Description
Technical field
The invention belongs to stem cells technology fields, are related to a kind of kit for being used to prepare umbilical cord mesenchymal stem cells.
Background technique
Source for mesenchymal stem cells is initially found in marrow in the mesoderm and ectoderm of mesoderm growing early stage, because it has
Multi-lineage potential, hematopoiesis support and promotes stem cell implantation, immunoregulation and be increasingly subject to people the features such as self-replacation
Concern can be divided into fat, bone, cartilage, muscle, tendon, ligament, mind in vivo or in vitro under specific inductive condition
Through Various Tissues cells such as, liver, cardiac muscle, endotheliums, still there is multi-lineage potential after continuous passage culture and freezen protective, have
Boundless potential applicability in clinical practice.
Mescenchymal stem cell is mainly derived from marrow, umbilical cord, fat etc..Because with age, stem cell population and
Proliferative capacity is remarkably decreased, and therefore, marrow and adipose-derived mescenchymal stem cell quantity and activity are affected, and obtain
Take marrow and and fat it is invasive, cause donor painful, it is also possible to lead to infection, bleeding and chronic ache etc..
Umbilical cord mesenchymal stem cells (Umbilical Cord Mesenchymal Stem Cells, UC-MSCs) refer to
Be present in one of neonatal umbilical cord tissue with self-renewing, multi-lineage potential stem cell, can be divided into heart,
The Various Tissues cell such as liver, kidney, lung, pancreas, nerve, bone, muscle, fat almost covers all histocytes of human body.
The organized block adherent method of the acquisition of umbilical cord mesenchymal stem cells and collagenase digestion.The tissue block adherent method originally culture time is
In or so two to three weeks, incubation time is longer, and the mesenchyma of a large amount of adherent growth can be quickly obtained using collagenase digestion
Stem cell, this method is simple and easy to do, may better maintain cell viability, substantially reduces the originally culture time.
Umbilical cord mesenchymal stem cells are with the following functions and purposes: 1, the histoorgan of damage or lesion are repaired, for controlling
Treat cardiovascular and cerebrovascular disease, hepatopathy, bone and muscle degenerative disease, brain and neurologic defict and senile dementia etc.；2, have compared with
Strong immunoregulation effect can be used for treating the autoimmune diseases such as lupus erythematosus and chorionitis, reduces cell or organ moves
Immunological rejection after plant improves the success rate of cell or organ transplant；3, promote Radiation in jury function, with single Hematopoietic Stem
Cell transplantation is compared, and mescenchymal stem cell and candidate stem cell co-transplantation can significantly improve the diseases such as leukaemia and refractory anemia
Therapeutic effect.
Umbilical cord mesenchymal stem cells have the advantage that mescenchymal stem cell rich content in 1, umbilical cord；2, it is filled between umbilical cord
Matter stem cells hyperplasia ability and differentiation capability are stronger；3, it can repeatedly expand, provide a large amount of seed cell for clinical treatment, foot
Needs are treated to meet；4, immunogenicity is low；5, extensive clinical application and huge medical prospect: to myocardial infarction, glycosuria
A variety of diseases such as disease, cirrhosis, lupus erythematosus, parkinsonism, senile dementia, spinal cord injury have certain therapeutic effect,
With scientific and technological progress, it can be used for the reparation and reconstruction of tissue and organ.Umbilical cord mesenchymal stem cells have extremely wide clinic
Application prospect.Therefore, it develops simple, quick, high-new, safety and obtains a large amount of umbilical cord mesenchymal stem cells methods as when business
It is anxious.
Summary of the invention
It is an object of the present invention to provide a benefits to be exclusively used in the kit that collagenase digestion extracts umbilical cord mesenchymal stem cells, benefit
A large amount of umbilical cord mesenchymal stem cells can be quickly obtained with the reagent in kit, it is easy to use, securely and reliably.
To achieve the above object, the technical solution adopted by the present invention is that: be used to prepare the reagent of umbilical cord mesenchymal stem cells
Box, umbilical cord preserving fluid, umbilical cord cleaning solution, umbilical cord decomposed solution, cell washing solution, cell culture fluid including bottled preservation respectively,
Cell dissociation buffer, cells frozen storing liquid.
The umbilical cord preserving fluid is containing 100~200ug/ml of penicillin, 100~200ug/ml of streptomysin, anphotericin 2.5
~5 μ g/ml, 2~3ng/ml of progesterone, 10~30ug/ml of insulin, 2~4nmol/ml of vitamin C DMEM, DMEM/F12,
MEM or RPMI-1640 culture medium；
The umbilical cord cleaning solution is containing 100~200ug/ml of penicillin, 100~200ug/ml of streptomysin, 2.5~5 μ of anphotericin
G/ml, 2~3ng/ml of progesterone, 10~30ug/ml of insulin, the physiological saline of 2~4nmol/ml of vitamin C, PBS, D-
Hanks or HBS；
The umbilical cord decomposed solution be containing II 1~2mg/ml of clostridiopetidase A, 0.1~0.2 mg/ml of deoxyribonuclease Ⅰ, progesterone 2~
DMEM, DMEM/F12, MEM or RPMI-1640 training of 3ng/ml, 10~30ug/ml of insulin, 2~4nmol/ml of vitamin C
Support base；
The cell washing solution is containing 2~3ng/ml of progesterone, 10~30ug/ml of insulin, 2~4nmol/ml of vitamin C
PBS, D-Hanks or HBSS；
The cell culture fluid is containing between 2~3ng/ml of progesterone, 10~30ug/ml of insulin, 2~4nmol/ml of vitamin C
Mesenchymal stem cells serum free growth medium, the culture medium chemical component limit, and contain L-Glutamine, are free of phenol red and antibiosis
Element；
The cell dissociation buffer be containing 1.25~2.5mg/ml of Trypsin-EDTA, containing 2~3ng/ml of progesterone, insulin 10~
PBS, D-Hanks or HBSS solution of 30ug/ml, 2~4nmol/ml of vitamin C；
The cells frozen storing liquid is to contain 2~3ng/ml of progesterone, 10~30ug/ml of insulin, dimension containing 60~200mg/ml of DMSO
Raw element C 2~4nmol/ml mesenchymal stem cell serum-free growth medium.
A kind of kit preparing umbilical cord mesenchymal stem cells provided by the invention, in the kit, umbilical cord preserving fluid
Effect is the activity guaranteed in the transportational process of umbilical cord sample after collection, and is eliminated and pre- bacteriological protection, fungi.Umbilical cord washing
The effect of liquid is the blood cell that washing removes umbilical cord tissue, while being eliminated and pre- bacteriological protection, fungi.The effect of umbilical cord decomposed solution
It is digestion umbilical cord tissue, obtains single cell suspension.The effect of cell washing solution is the stem cell that washing obtains.Cell culture fluid is
Serum free growth medium, and chemical component limits, and reduces the pathogenic infection risk of animal origin, culture umbilical cord mesenchyma is dry
Cell is safer for using fetal calf serum.The effect of cell dissociation buffer be cell passage when, digest it is adherent between fill
Matter stem cell.The effect of cells frozen storing liquid is the umbilical cord mesenchymal stem cells for freezing acquisition.
The kit for being used to prepare umbilical cord mesenchymal stem cells of the invention can obtain a large amount of in short period (in 2 weeks)
Umbilical cord mesenchymal stem cells, cell quantity is up to 1010The order of magnitude, and there is easy to use, low in cost, serum-free, it reduces
The pathogenic infection risk of animal origin, it is safer the features such as, will be played in stem cell clinical test and its correlative study weight
It acts on, 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 application of the kit of umbilical cord mesenchymal stem cells
One, it is used to prepare the preparation of the kit of umbilical cord mesenchymal stem cells
1, solution allocation
The kit for being used to prepare umbilical cord mesenchymal stem cells of the invention includes following reagent:
(1) umbilical cord preserving fluid configuration method: penicillin (brand Amresco, article No. 0242) 200mg, streptomysin (brand
Amresco, article No. 0382) 200mg, anphotericin (brand Amresco, article No. E437) 5mg, progesterone (Sigma, V900699-
5g) 2 μ g, insulin (Sigma, I0908) 10mg, vitamin C (Sigma, A5960-10mg) 2mmol, are dissolved in 1000ml
0.22um filtration sterilization in DMEM culture medium.
(2) umbilical cord cleaning solution configuration method: penicillin 200mg, streptomysin 200mg, anphotericin 5mg, 2 μ g of progesterone, pancreas
Island element 10mg, vitamin C 2mmol are dissolved in 0.22um filtration sterilization in 1000ml physiological saline.
(3) umbilical cord decomposed solution configuration method: clostridiopetidase A II (brand sigma, article No. C6885) 1 g, DNA
Enzyme I (brand sigma, article No. C4263) 0.1g, 2 μ g of progesterone, insulin 10mg, vitamin C 2mmol are dissolved in 1000ml DMEM
In culture medium, 0.22um filter filtration sterilization.
(4) cell washing solution configuration method: NaCl 8.50g, Na2HPO4·12H2O 3.58g、NaH2PO4·2H2O
0.39g adds distilled water to 1000ml, adjusts pH between 7.2-7.4, high pressure sterilization is up to PBS solution；2 μ g of progesterone, pancreas islet
Plain 10mg, vitamin C 2mmol are dissolved in above-mentioned configured 1000ml PBS solution, 0.22um filter filtration sterilization.
(5) cell culture fluid configuration method: serum-free basal medium (brand Lonza, article No. 00190620) and people
Growth of mesenchymal stem cells serum free medium cell factor (brand Lonza, article No. 00192125) is with the volume ratio of 9:1
Mixing prepares serum free medium；2 μ g of progesterone, insulin 10mg, vitamin C 2mmol are dissolved in above-mentioned configured
In 1000ml serum-free basal medium, 0.22um filter filtration sterilization.
(6) cell dissociation buffer configuration method: Trypsin(trypsase) 0.25g, EDTA-2Na 19.728mg be dissolved in
It states in the cell washing solution 100ml prepared, mixes, 0.22um filter filtration sterilization.
(7) cells frozen storing liquid configuration method: 10ml DMSO(brand sigma, article No. D2650) it is slowly added on 90ml
The cell culture fluid prepared is stated, is mixed 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: 1 bottle of umbilical cord preserving fluid (50ml/ bottles), umbilical cord cleaning solution
1 bottle (50ml/ bottles), 1 bottle of umbilical cord decomposed solution (50ml/ bottles), 5 bottles of cell washing solution (1000ml/ bottles), 10 bottles of cell culture fluid
(1000ml/ bottles), 2 bottles of cell dissociation buffer (1000ml/ bottles), 2 bottles of cells frozen storing liquid (1000ml/ bottles).
Each component in kit is dispensed according to mentioned reagent amount, is packaged to be that be used to prepare umbilical cord mesenchyma 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
4 DEG C of preservations later are finished in one month.
Two, the preparation of umbilical cord mesenchymal stem cells
Current the present embodiment kit prepares umbilical cord mesenchymal stem cells, and all maneuvering sequences are completed in clean bench, tool
Preparation includes the following steps, operates by taking 5cm umbilical cord as an example as follows:
1, transport umbilical cord: isometric umbilical cord preserving fluid is added in acquisition umbilical cord 5cm, and 2~8 DEG C of constant temperature are stored in vaccine box,
It is sent in 48h to laboratory.
2, it washs umbilical cord: inhaling and abandon umbilical cord preserving fluid, isometric umbilical cord cleaning solution is added, vibrates several times repeatedly, until umbilical cord is washed
Wash liquid clarification.
3, umbilical cord is decomposed:
1) it takes umbilical cord to be placed in 10cm culture dish, is cut into several sections, and reject intermediate three blood vessels；
2) white connective tissue between amnion and blood vessel is Wal Tong Shi glue, is torn, is put with long handle pincers
Enter in sterilized petri dishes；
3) with sterile long handle operating scissors, Wal Tong Shi glue is cut into 1mm in centrifuge tube3The tissue of left and right is homogenized block, shearing
15~20min of time；
4) be added with the isometric umbilical cord decomposed solution of tissue homogenate block, be preheating to 37 DEG C, be placed in constant-temperature shaking incubator, 37 DEG C,
200rpm digests 60~90min, and every 20min taking-up is put back to after gently shaking up, until umbilical cord tissue homogenate block is in waste shape.
4, it collects cell: the tissue after enzymic digestion being homogenized according to volume ratio 1:6, the cell washing solution of 6 times of volumes is added,
It is filtered to remove residue by sterile 200 mesh filter screen, 1ml suspension is drawn and counts cell, is 5.1 × 107It is a, remaining liquid
400g is centrifuged 8min, abandons supernatant, obtains lower confluent monolayer cells.
5, cell seeding: according to cell count, according to 1.0 × 106Cell culture fluid 50ml is added in the density of a/ml, connects
Kind of 3 T175 culture bottles, are placed in carbon dioxide incubator, condition of culture: 37 ± 0.5 DEG C, carbon dioxide volume fraction be (5 ±
0.2) % is changed the liquid once every 3d.
6, cell passes on: 4~5d or so, and primary cultured cell is inhaled up to when 70%~80% fusion and abandons old cell culture fluid, add
Cell washing solution stands 1min, inhales and abandons cell washing solution, and cell dissociation buffer is added, and digestion time is 1.5~2.5min, then
2~3ml of cell culture fluid is added and stops digestion, blows and beats bottom of bottle repeatedly and largely falls off to cell, move into 50ml centrifuge tube, carefully
Born of the same parents count, and are 1.5 × 108A, 400g is centrifuged 8min, abandons supernatant, obtains 1.5 × 108A P0 is for umbilical cord mesenchymal stem cells.
According to 5.0 × 105Cell culture fluid is added in a/ml, is inoculated into 15 T175 culture bottles, is put into training in carbon dioxide incubator
It supports, is calculated as P1 generation.It, up to when 80%~90% fusion, is ibid operated to 3d or so, P1 for cell, harvest cell, counting acquisition 7.8 ×
108A P1 ibid continues to pass on (every 3~4d can be passed on once) for umbilical cord mesenchymal stem cells, until harvest P3 generation, through cell
It is counted as 2.1 × 1010A umbilical cord mesenchymal stem cells.
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 by a small amount of umbilical cord tissue (long 5cm), being obtained within a short period of time (2 weeks) with kit of the invention
Obtain a large amount of umbilical cord mesenchymal stem cells (2.1 × 1010), show that kit of the invention can obtain largely within a short period of time
Umbilical cord mesenchymal stem cells.
Embodiment two is used to prepare the preparation and application of the kit of umbilical cord mesenchymal stem cells
One, it is used to prepare the preparation of the kit of umbilical cord mesenchymal stem cells
1, solution allocation
The kit for being used to prepare umbilical cord mesenchymal stem cells of the invention includes following reagent:
(1) umbilical cord preserving fluid configuration method: penicillin 100mg, streptomysin 100mg, anphotericin 2.5mg, 3 μ g of progesterone, pancreas islet
Plain 30mg, vitamin C 4mmol are dissolved in 1000ml DMEM/F12 culture medium 0.22um filtration sterilization packing.
(2) umbilical cord cleaning solution configuration method: penicillin 100mg, streptomysin 100mg, anphotericin 2.5mg, 3 μ g of progesterone,
Insulin 3 0mg, vitamin C 4mmol are dissolved in 1000ml physiological saline 0.22um filtration sterilization packing.
(3) umbilical cord decomposed solution configuration method: II 2 g of clostridiopetidase A, deoxyribonuclease Ⅰ 0.2g, 3 μ g of progesterone, pancreas islet
Plain 30mg, vitamin C 4mmol are dissolved in 1000ml DMEM/F12 culture medium, 0.22um filter filtration sterilization.
(4) cell washing solution configuration method: preparing PBS first, is formulated as NaCl 8.50g, Na2HPO4·12H2O
3.58g、NaH2PO4·2H2O 0.39g adds distilled water to 1000ml, adjusts pH between 7.2-7.4, high pressure sterilization to obtain the final product
PBS solution.3 μ g of progesterone, Insulin 3 0mg, vitamin C 4mmol are dissolved in above-mentioned configured 1000ml PBS solution,
0.22um filter filtration sterilization.
(5) cell culture fluid configuration method: serum-free basal medium and human mesenchymal stem cell grow free serum culture
Basal cell's factor has been mixed with serum free medium with the volume ratio of 9:1；3 μ g of progesterone, Insulin 3 0mg, vitamin C
4mmol is dissolved in above-mentioned configured 1000ml serum free medium, 0.22um filter filtration sterilization.
(6) cell dissociation buffer configuration method: Trypsin 125mg, EDTA-2Na 9.864mg be dissolved in it is above-mentioned prepare it is thin
In born of the same parents' cleaning solution 100ml, mix, 0.22um filter filtration sterilization.
(7) cells frozen storing liquid configuration method: 10ml DMSO is slowly added into the above-mentioned cell culture fluid prepared of 90ml,
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: 1 bottle of umbilical cord preserving fluid (50ml/ bottles), umbilical cord cleaning solution
1 bottle (50ml/ bottles), 1 bottle of umbilical cord decomposed solution (50ml/ bottles), 5 bottles of cell washing solution (1000ml/ bottles), 10 bottles of cell culture fluid
(1000ml/ bottles), 2 bottles of cell dissociation buffer (1000ml/ bottles), 2 bottles of cells frozen storing liquid (1000ml/ bottles)
Each component in kit is dispensed according to mentioned reagent amount, is packaged to be and is used to prepare umbilical cord mesenchymal stem cells
Kit.- 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, 4 after defrosting
It DEG C saves, is finished in one month.
Two, the preparation of umbilical cord mesenchymal stem cells
With the kit of step 1, method prepares umbilical cord mesenchymal stem cells in reference implementation example one, and with identical method pair
The mescenchymal stem cell of acquisition is counted, as a result 5cm umbilical cord, and amplification obtains 2.8 × 10 within 2 weeks10A umbilical cord mesenchyma is dry
Cell shows that kit of the invention can obtain a large amount of umbilical cord mesenchymal stem cells within a short period of time.
Embodiment three is used to prepare the preparation and application of the kit of umbilical cord mesenchymal stem cells
One, it is used to prepare the preparation of the kit of umbilical cord mesenchymal stem cells
1, solution allocation
The kit for being used to prepare umbilical cord mesenchymal stem cells of the invention includes following reagent:
(1) umbilical cord preserving fluid configuration method: penicillin 150mg, streptomysin 150mg, anphotericin 2.5mg, 2.5 μ g of progesterone, pancreas
Island element 25mg, vitamin C 3mmol are dissolved in 1000ml DMEM culture medium 0.22um filtration sterilization packing.
(2) umbilical cord cleaning solution configuration method: penicillin 150mg, streptomysin 150mg, anphotericin 2.5mg, 2.5 μ of progesterone
G, insulin 25mg, vitamin C 3mmol are dissolved in 1000ml physiological saline 0.22um filtration sterilization.
(3) umbilical cord decomposed solution configuration method: II 2 g of clostridiopetidase A, deoxyribonuclease Ⅰ 0.2g, 2.5 μ g of progesterone, pancreas
Island element 25mg, vitamin C 3mmol are 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, adjusts pH between 7.2-7.4, high pressure sterilization.2.5 μ g of progesterone,
Insulin 25mg, vitamin C 3mmol are dissolved in above-mentioned 1000ml PBS culture medium, 0.22um filter filtration sterilization.
(5) cell culture fluid configuration method: serum-free basal medium 450ml and its sterile mixing of additive 50ml.It is pregnant
2.5 μ g of ketone, insulin 25mg, vitamin C 3mmol are dissolved in above-mentioned 1000ml serum-free basal medium, 0.22um filter mistake
Filter out bacterium.
(6) cell dissociation buffer configuration method: Trypsin 0.25g, EDTA-2Na 19.728mg be dissolved in it is above-mentioned prepare it is thin
In born of the same parents' cleaning solution 100ml, mix, 0.22um filter filtration sterilization.
(7) cells frozen storing liquid configuration method: 15ml DMSO is slowly added into the above-mentioned cell culture fluid prepared of 85ml,
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: 1 bottle of umbilical cord preserving fluid (50ml/ bottles), umbilical cord cleaning solution
1 bottle (50ml/ bottles), 1 bottle of umbilical cord decomposed solution (50ml/ bottles), 5 bottles of cell washing solution (1000ml/ bottles), 10 bottles of cell culture fluid
(1000ml/ bottles), 2 bottles of cell dissociation buffer (1000ml/ bottles), 2 bottles of cells frozen storing liquid (1000ml/ bottles)
Each component in kit is dispensed according to mentioned reagent amount, is packaged to be and is used to prepare umbilical cord mesenchymal stem cells
Kit.- 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, 4 after defrosting
It DEG C saves, is finished in one month.
Two, the preparation of umbilical cord mesenchymal stem cells
With the kit of step 1, method prepares umbilical cord mesenchymal stem cells in reference implementation example one, and with identical method pair
The mescenchymal stem cell of acquisition is counted, as a result 5cm umbilical cord, and amplification obtains 2.2 × 10 within 2 weeks10A umbilical cord mesenchyma is dry
Cell shows that kit of the invention can obtain a large amount of umbilical cord mesenchymal stem cells within a short period of time.
Example IV is used to prepare the preparation and application of the kit of umbilical cord mesenchymal stem cells
One, it is used to prepare the preparation of the kit of umbilical cord mesenchymal stem cells
1, solution allocation
The kit for being used to prepare umbilical cord mesenchymal stem cells of the invention includes following reagent:
(1) umbilical cord preserving fluid configuration method: penicillin 200mg, streptomysin 200mg, anphotericin 5mg, 2 μ g of progesterone, insulin
25mg, vitamin C 4mmol are dissolved in 0.22um filtration sterilization in 1000ml RPMI-1640 culture medium and dispense.
(2) umbilical cord cleaning solution configuration method: penicillin 200mg, streptomysin 200mg, anphotericin 5mg, 2 μ g of progesterone, pancreas
Island element 25mg, vitamin C 4mmol are dissolved in 1000ml physiological saline 0.22um filtration sterilization packing.
(3) umbilical cord decomposed solution configuration method: II 1.5 g of clostridiopetidase A, deoxyribonuclease Ⅰ 0.15g, 2 μ g of progesterone,
Insulin 25mg, vitamin C 4mmol are dissolved in 1000ml RPMI-1640 culture medium, 0.22um filter filtration sterilization.
(4) cell washing solution configuration method: 8.0 0.4 g of g, KCl of 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 is gone out
Bacterium prepares HBSS.2 μ g of progesterone, insulin 25mg, vitamin C 4mmol are dissolved in the 1000ml HBSS prepared, 0.22um
Filter filtration sterilization.
(5) cell culture fluid configuration method: serum-free basal medium 450ml and its sterile mixing of additive 50ml.It is pregnant
2 μ g of ketone, insulin 25mg, vitamin C 4mmol are dissolved in the 1000ml serum-free basal medium prepared, 0.22um filter mistake
Filter out bacterium.
(6) cell dissociation buffer configuration method: Trypsin 0.25g, EDTA-2Na 19.728mg be dissolved in it is above-mentioned prepare it is thin
In born of the same parents' cleaning solution 100ml, mix, 0.22um filter filtration sterilization.
(7) cells frozen storing liquid configuration method: 10ml DMSO is slowly added into the above-mentioned cell culture fluid prepared of 90ml,
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: 1 bottle of umbilical cord preserving fluid (50ml/ bottles), umbilical cord cleaning solution
1 bottle (50ml/ bottles), 1 bottle of umbilical cord decomposed solution (50ml/ bottles), 5 bottles of cell washing solution (1000ml/ bottles), 10 bottles of cell culture fluid
(1000ml/ bottles), 2 bottles of cell dissociation buffer (1000ml/ bottles), 2 bottles of cells frozen storing liquid (1000ml/ bottles)
Each component in kit is dispensed according to mentioned reagent amount, is packaged to be and is used to prepare umbilical cord mesenchymal stem cells
Kit.- 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, 4 after defrosting
It DEG C saves, is finished in one month.
Two, the preparation of umbilical cord mesenchymal stem cells
With the kit of step 1, method prepares umbilical cord mesenchymal stem cells in reference implementation example one, and with identical method pair
The mescenchymal stem cell of acquisition is counted, as a result 5cm umbilical cord, and amplification obtains 2.9 × 10 within 2 weeks10A umbilical cord mesenchyma is dry
Cell shows that kit of the invention can obtain a large amount of umbilical cord mesenchymal stem cells within a short period of time.
Embodiment five is used to prepare the preparation and application of the kit of umbilical cord mesenchymal stem cells
One, it is used to prepare the preparation of the kit of umbilical cord mesenchymal stem cells
1, solution allocation
The kit for being used to prepare umbilical cord mesenchymal stem cells of the invention includes following reagent
(1) umbilical cord preserving fluid configuration method: penicillin 200mg, streptomysin 200mg, anphotericin 5mg, 2.8 μ g of progesterone, pancreas islet
Plain 12mg, vitamin C 3.2mmol are dissolved in 1000ml DMEM culture medium, 0.22um filtration sterilization, packing.
(2) umbilical cord cleaning solution configuration method: penicillin 200mg, streptomysin 200mg, anphotericin 5mg, 2.8 μ g of progesterone,
Insulin 12mg, vitamin C 3.2mmol, are dissolved in 1000ml physiological saline, 0.22um filtration sterilization, packing.
(3) umbilical cord decomposed solution configuration method: II 1.5 g of clostridiopetidase A, deoxyribonuclease Ⅰ 0.15g, 2.8 μ g of progesterone,
Insulin 12mg, vitamin C 3.2mmol are dissolved in 1000ml DMEM culture medium, 0.22um filter filtration sterilization.
(4) cell washing solution configuration method: D-Hanks culture medium, KCl 0.40g, KH are prepared first2PO4 0.06g、
NaCl 8.00g、NaHCO3 0.35g、Na2HPO4·12H2O 0.12g adds tri-distilled water to 1000ml, adjusts pH to 7.2-7.4
Between, high pressure sterilization；Then 2.8 μ g of progesterone, insulin 12mg, vitamin C 3.2mmol are dissolved in the 1000ml prepared
In D-Hanks culture medium, 0.22um filter filtration sterilization.
(5) cell culture fluid configuration method: serum-free basal medium 450ml and its sterile mixing of additive 50ml.It is pregnant
2.8 μ g of ketone, insulin 12mg, vitamin C 3.2mmol are dissolved in the 1000ml serum-free basal medium prepared, 0.22um filter
Device filtration sterilization.
(6) cell dissociation buffer configuration method: Trypsin 0.25g, EDTA-2Na 19.728mg be dissolved in it is above-mentioned prepare it is thin
In born of the same parents' cleaning solution, mix, 0.22um filter filtration sterilization.
(7) cells frozen storing liquid configuration method: 10ml DMSO is slowly added into the above-mentioned cell culture fluid prepared of 90ml,
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: 1 bottle of umbilical cord preserving fluid (50ml/ bottles), umbilical cord cleaning solution
1 bottle (50ml/ bottles), 1 bottle of umbilical cord decomposed solution (50ml/ bottles), 5 bottles of cell washing solution (1000ml/ bottles), 10 bottles of cell culture fluid
(1000ml/ bottles), 2 bottles of cell dissociation buffer (1000ml/ bottles), 2 bottles of cells frozen storing liquid (1000ml/ bottles)
Each component in kit is dispensed according to mentioned reagent amount, is packaged to be and is used to prepare umbilical cord mesenchymal stem cells
Kit.- 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, 4 after defrosting
It DEG C saves, is finished in one month.
Two, the preparation of umbilical cord mesenchymal stem cells
With the kit of step 1, method prepares umbilical cord mesenchymal stem cells in reference implementation example one, and with identical method pair
The mescenchymal stem cell of acquisition is counted, as a result 5cm umbilical cord, and amplification obtains 2.4 × 10 within 2 weeks10A umbilical cord mesenchyma is dry
Cell shows that kit of the invention can obtain a large amount of umbilical cord mesenchymal stem cells within a short period of time.
Claims (9)
1. the kit of umbilical cord mesenchymal stem cells is used to prepare, it is characterized in that: including the umbilical cord guarantor of bottled preservation respectively in box
Liquid storage, umbilical cord cleaning solution, umbilical cord decomposed solution, cell washing solution, cell culture fluid, cell dissociation buffer, cells frozen storing liquid.
2. according to the kit according to claim 1 for being used to prepare umbilical cord mesenchymal stem cells, it is characterized in that: the examination
The amount of each component in agent box are as follows:
1 bottle of umbilical cord preserving fluid, 50ml/ bottles；
1 bottle of umbilical cord cleaning solution, 50ml/ bottles；
1 bottle of umbilical cord decomposed solution, 50ml/ bottles；
5 bottles of cell washing solution, 1000ml/ bottles；
10 bottles of cell culture fluid, 1000ml/ bottles；
2 bottles of cell dissociation buffer, 1000ml/ bottles；
2 bottles of cells frozen storing liquid, 1000ml/ bottles.
3. the kit according to claim 1 or 2 for being used to prepare umbilical cord mesenchymal stem cells, it is characterized in that: the navel
It is containing 100~200ug/ml of penicillin, 100~200ug/ml of streptomysin, 2.5~5 μ g/ml of anphotericin, progesterone 2 that band, which saves liquid,
DMEM, DMEM/F12, MEM or RPMI-1640 of~3ng/ml, 10~30ug/ml of insulin, 2~4nmol/ml of vitamin C
Culture medium.
4. the kit according to claim 1 or 2 for being used to prepare umbilical cord mesenchymal stem cells, it is characterized in that: the navel
Band cleaning solution is containing 100~200ug/ml of penicillin, 100~200ug/ml of streptomysin, 2.5~5 μ g/ml of anphotericin, progesterone 2
~3ng/ml, 10~30ug/ml of insulin, the physiological saline of 2~4nmol/ml of vitamin C, PBS, D-Hanks or HBS.
5. the kit according to claim 1 or 2 for being used to prepare umbilical cord mesenchymal stem cells, it is characterized in that: the navel
Band decomposed solution is containing II 1~2mg/ml of clostridiopetidase A, 0.1~0.2 mg/ml of deoxyribonuclease Ⅰ, 2~3ng/ml of progesterone, pancreas
DMEM, DMEM/F12, MEM or RPMI-1640 culture medium of island 10~30ug/ml of element, 2~4nmol/ml of vitamin C.
6. the kit according to claim 1 or 2 for being used to prepare umbilical cord mesenchymal stem cells, it is characterized in that: described thin
Born of the same parents' cleaning solution is PBS, D-Hanks containing 2~3ng/ml of progesterone, 10~30ug/ml of insulin, 2~4nmol/ml of vitamin C
Or HBSS.
7. the kit according to claim 1 or 2 for being used to prepare umbilical cord mesenchymal stem cells, it is characterized in that: described thin
Born of the same parents' culture solution is the mescenchymal stem cell containing 2~3ng/ml of progesterone, 10~30ug/ml of insulin, 2~4nmol/ml of vitamin C
Serum free growth 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 umbilical cord mesenchymal stem cells, it is characterized in that: described thin
Born of the same parents' digestive juice is to contain 2~3ng/ml of progesterone, 10~30ug/ml of insulin, dimension containing 1.25~2.5mg/ml of Trypsin-EDTA
PBS, D-Hanks or HBSS solution of raw 2~4nmol/ml of element C.
9. the kit according to claim 1 or 2 for being used to prepare umbilical cord mesenchymal stem cells, it is characterized in that: described thin
Born of the same parents' frozen stock solution be containing 60~200mg/ml of DMSO, containing 2~3ng/ml of progesterone, 10~30ug/ml of insulin, vitamin C 2~
4nmol/ml mesenchymal stem cell serum-free growth medium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710718154.8A CN109423477A (en) | 2017-08-21 | 2017-08-21 | It is used to prepare the kit of umbilical cord mesenchymal stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710718154.8A CN109423477A (en) | 2017-08-21 | 2017-08-21 | It is used to prepare the kit of umbilical cord mesenchymal stem cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109423477A true CN109423477A (en) | 2019-03-05 |
Family
ID=65497997
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710718154.8A Pending CN109423477A (en) | 2017-08-21 | 2017-08-21 | It is used to prepare the kit of umbilical cord mesenchymal stem cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109423477A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109897818A (en) * | 2019-03-20 | 2019-06-18 | 江苏瑞思坦生物科技有限公司 | A kind of umbilical cord tissue cleaning solution and preparation method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103243071A (en) * | 2013-05-09 | 2013-08-14 | 陈云燕 | Clinical-grade human mesenchymal stem cell serum-free complete medium |
| CN103589683A (en) * | 2013-08-12 | 2014-02-19 | 北京东方华辉生物医药科技有限公司 | Separation method and culture method for umbilical cord mesenchymal stem cells |
| CN104232572A (en) * | 2014-08-20 | 2014-12-24 | 北京瑞思德生物科技有限公司 | Kit for preparing umbilical cord mesenchymal stem cells |
| CN104928238A (en) * | 2014-03-21 | 2015-09-23 | 青岛中天干细胞工程有限公司 | Adipose mesenchymal stem cell preparation kit |
| CN106554939A (en) * | 2015-09-24 | 2017-04-05 | 周宇璠 | Human umbilical cord mesenchymal stem cells are separated and immunosuppressant can strengthen cultivate reagent box |
-
2017
- 2017-08-21 CN CN201710718154.8A patent/CN109423477A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103243071A (en) * | 2013-05-09 | 2013-08-14 | 陈云燕 | Clinical-grade human mesenchymal stem cell serum-free complete medium |
| CN103589683A (en) * | 2013-08-12 | 2014-02-19 | 北京东方华辉生物医药科技有限公司 | Separation method and culture method for umbilical cord mesenchymal stem cells |
| CN104928238A (en) * | 2014-03-21 | 2015-09-23 | 青岛中天干细胞工程有限公司 | Adipose mesenchymal stem cell preparation kit |
| CN104232572A (en) * | 2014-08-20 | 2014-12-24 | 北京瑞思德生物科技有限公司 | Kit for preparing umbilical cord mesenchymal stem cells |
| CN106554939A (en) * | 2015-09-24 | 2017-04-05 | 周宇璠 | Human umbilical cord mesenchymal stem cells are separated and immunosuppressant can strengthen cultivate reagent box |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109897818A (en) * | 2019-03-20 | 2019-06-18 | 江苏瑞思坦生物科技有限公司 | A kind of umbilical cord tissue cleaning solution and preparation method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR100779812B1 (en) | Preservation of non embryonic cells from non hematopoietic tissues | |
| Toupadakis et al. | Comparison of the osteogenic potential of equine mesenchymal stem cells from bone marrow, adipose tissue, umbilical cord blood, and umbilical cord tissue | |
| KR100494265B1 (en) | Composition for treatment of articular cartilage damage | |
| Hernigou et al. | Stem cell therapy for the treatment of hip osteonecrosis: a 30-year review of progress | |
| Hernigou et al. | The use of percutaneous autologous bone marrow transplantation in nonunion and avascular necrosis of bone | |
| US7060494B2 (en) | Growth of human Mesenchymal Stem Cells (hMSC) using umbilical cord blood serum and the method for the preparation thereof | |
| WO2019161591A1 (en) | Isolation and cultivation method for mesenchymal stem cells, as well as cryopreservation and resuscitation method for same | |
| Homma et al. | Cellular therapies for the treatment of non-union: the past, present and future | |
| AU2002326901A1 (en) | Preservation of non embryonic cells from non hematopoietic tissues | |
| CZ200649A3 (en) | Method of culturing human mezenchymal stem cells, particularly for facilitating fracture healing process, and bioreactor for carrying out the method | |
| EP2083755A1 (en) | Medical kit and using method thereof | |
| CN104928238A (en) | Adipose mesenchymal stem cell preparation kit | |
| US11938247B2 (en) | Human nasal turbinate-derived mesenchymal stem cell-based, 3D bioprinted construct, and use thereof | |
| US20090016997A1 (en) | Autologous/allogeneic human DNA grafting, anti-and reverse aging stem cell, and bone marrow compositions/methods | |
| CN104983742A (en) | Stem cell preparation for treating degenerative osteoarthropathy and preparation method of stem cell preparation | |
| US9956317B2 (en) | Clinical applications of formulations containing adipose-derived stem cells | |
| CN104232572A (en) | Kit for preparing umbilical cord mesenchymal stem cells | |
| US9434923B2 (en) | Preparation of parental cell bank from foetal tissue | |
| CN109423476A (en) | It is used to prepare the kit of fat mesenchymal stem cell | |
| CN109423477A (en) | It is used to prepare the kit of umbilical cord mesenchymal stem cells | |
| CN110923202A (en) | Material and material box for separating, culturing and storing dental pulp mesenchymal stem cells | |
| de Buys Roessingh et al. | Cell therapy assistance in reconstructive surgery for musculoskeletal tissues following burn and trauma: Swiss cellular Transplantation Platform | |
| CN110373381A (en) | A kind of preparation method by the efficient placenta mesenchyma stem cell of homogenizer | |
| US20160130557A1 (en) | Formulations Containing and Kit for Using Adipose-Derived Stem Cells and Use Thereof | |
| Ruiz‐Lopez et al. | Complications in Regenerative Medicine |
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 | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20190305 |