CN110199984A - A kind of umbilical cord mesenchymal stem cells frozen stock solution and application thereof - Google Patents
A kind of umbilical cord mesenchymal stem cells frozen stock solution and application thereof Download PDFInfo
- Publication number
- CN110199984A CN110199984A CN201910451040.0A CN201910451040A CN110199984A CN 110199984 A CN110199984 A CN 110199984A CN 201910451040 A CN201910451040 A CN 201910451040A CN 110199984 A CN110199984 A CN 110199984A
- Authority
- CN
- China
- Prior art keywords
- cell
- surface antigen
- umbilical cord
- stem cells
- mesenchymal stem
- 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
-
- 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
-
- 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
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Biophysics (AREA)
- Physiology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of umbilical cord mesenchymal stem cells frozen stock solutions and application thereof, frozen stock solution includes human serum albumin solution, dimethyl sulphoxide solution, trophic factors and pharmaceutically acceptable carrier, the trophic factors is one of blood vessel endothelial cell growth factor VEGF, transforming growth factor TGF, interleukin 6 IL-6, fibrous bud Porcine HGF bFGF, hepatocyte growth factor HGF or a variety of, application of the umbilical cord mesenchymal stem cells frozen stock solution in umbilical cord mesenchymal stem cells freeze.By the above-mentioned means, the present invention with freezing and storing umbilical mesenchymal stem cells and can be made the umbilical cord mesenchymal stem cells after freezing keep activity and differentiation stemness, be frozen effect with good using the specific frozen stock solution of one kind.
Description
Technical field
The present invention relates to cell cryopreservation technical fields, more particularly to a kind of umbilical cord mesenchymal stem cells frozen stock solution and its use
On the way.
Background technique
Cell cryopreservation is that cell is placed on to low temperature environment, cell metabolism is reduced, so as to a kind of technology of long term storage.Cell
Freezing is one of main method that cell saves, plays the role of cell conservation.
Cell cryopreservation is that cell is placed in cryo-conservation in -196 DEG C of liquid nitrogen using Cryopreservation Technology, and cell can be made temporary
It is detached from growth conditions and saves its cell characteristics, recovery cell is used for clinical application again when needed in this way.It is suitable
Degree ground saves a certain amount of cell, can prevent from losing cell because the cell cultivated is contaminated or other accidents
Kind, play the role of cell conservation.In addition to this it is possible to be bought using the form of cell cryopreservation, post and give, exchange and transport
Send certain cells.Frozen stock solution is added when cell cryopreservation, freezing point of solution can be made to reduce, in addition under the conditions of slow freezing, into the cell
Moisture appears, and reduces ice crystal and is formed, to avoid cellular damage.Method using " slow freeze is melted fastly " can preferably guarantee cell
Survival.
Traditional cells frozen storing liquid contains basal medium, dimethyl sulfoxide, fetal calf serum etc. at present, but fetal calf serum at
Divide complexity, containing a large amount of alloantigen, is difficult to clean up in subsequent recovery culture link, feeds back to human body and cause antigen
Antibody side reaction, therefore tend to the substitute freeze-stored cell using serum-free at present.But for mescenchymal stem cell and
Speech, even if will also result in " the low temperature dizziness effect of mescenchymal stem cell after freezing using the frozen stock solution of serum-free substitute
(Cryo-stun effect) " leads to mescenchymal stem cell disability effect, loses " the stemness of mescenchymal stem cell
(stemness) ", it is extremely limited the clinical application of mescenchymal stem cell.
Summary of the invention
It is described the invention mainly solves the technical problem of providing a kind of umbilical cord mesenchymal stem cells frozen stock solution and application thereof
Umbilical cord mesenchymal stem cells frozen stock solution applies convenient, constituent optimization, is able to maintain and freezes rear umbilical cord mesenchymal stem cells " stemness
stemness”。
In order to solve the above technical problems, one technical scheme adopted by the invention is that: it is dry thin to provide a kind of umbilical cord mesenchyma
Born of the same parents' frozen stock solution, including human serum albumin solution, dimethyl sulphoxide solution, trophic factors and pharmaceutically acceptable carrier, institute
Stating trophic factors is blood vessel endothelial cell growth factor VEGF, transforming growth factor TGF, interleukin 6 IL-6, the life of fiber sprout cell
One of long factor bFGF, hepatocyte growth factor HGF or a variety of.
In a preferred embodiment of the present invention, the human serum albumin solution is people's blood that mass fraction is 5-15%
Pure protein solution；The dimethyl sulphoxide solution is the dimethyl sulphoxide solution that mass fraction is 5-10%；It is described intravascular
The concentration of skin cell growth factor is 2-3ng/mL, and the concentration of the fibrous bud Porcine HGF is 2-6ng/mL, described white
The concentration of interleukin 6 is 2-3ng/mL, and the concentration of the hepatocyte growth factor is 15-20ng/mL, the transforming growth factor
Concentration is 10-15ng/mL；The mass ratio of the human serum albumin solution and the dimethyl sulphoxide solution is 1-2:1；It is described
Pharmaceutically acceptable carrier is Multiple electrolytes injection；The umbilical cord mesenchymal stem cells frozen stock solution is unit dosage form, institute
The volume for stating unit dosage form is≤4mL.
In a preferred embodiment of the present invention, the human serum albumin solution is people's blood that mass fraction is 8-12%
Pure protein solution；The dimethyl sulphoxide solution is the dimethyl sulphoxide solution that mass fraction is 7-9%；The human seralbumin
The mass ratio of protein solution and the dimethyl sulphoxide solution is 1-1.5:1；The volume of the unit dosage form is≤2mL.
A kind of purposes of umbilical cord mesenchymal stem cells frozen stock solution is provided, the umbilical cord mesenchymal stem cells frozen stock solution is in umbilical cord
Application in mesenchymal stem cell cryopreserving.
In a preferred embodiment of the present invention, every 105-108Umbilical cord mesenchymal stem cells described in/mL apply each unit
The umbilical cord mesenchymal stem cells frozen stock solution.
In a preferred embodiment of the present invention, every 106-107Umbilical cord mesenchymal stem cells described in/mL apply each unit
The umbilical cord mesenchymal stem cells frozen stock solution.
In a preferred embodiment of the present invention, the cryopreservation methods are the umbilical cord mesenchymal stem cells with -1 DEG C/minute
The rate of clock is at the uniform velocity cooled to -80 DEG C, is subsequently placed in liquid nitrogen container and saves for a long time.
In a preferred embodiment of the present invention, umbilical cord mesenchymal stem cells after freezing have it is selected from the group below any or
Various features:
(i) 92% or more cell has surface antigen CD90；
(ii) 85% or more cell has surface antigen CD73；
(iii) 92% or more cell has surface antigen CD29；
(iv) 85% or more cell has surface antigen CD49d；
In a preferred embodiment of the present invention, umbilical cord mesenchymal stem cells after freezing have it is selected from the group below any or
Various features:
(i) 95% or more cell has surface antigen CD90；
(ii) 90% or more cell has surface antigen CD73；
(iii) 95% or more cell has surface antigen CD29；
(iv) 90% or more cell has surface antigen CD49d；
In a preferred embodiment of the present invention, umbilical cord mesenchymal stem cells after freezing have it is selected from the group below any or
Various features:
(i) 96% or more cell has surface antigen CD90；
(ii) 92% or more cell has surface antigen CD73；
(iii) 98% or more cell has surface antigen CD29；
(iv) 95% or more cell has surface antigen CD49d.
In a preferred embodiment of the present invention, mescenchymal stem cell after freezing have selected from the group below one or more
Feature:
(v) 2% cell below has surface antigen CD34；
(vi) 2% cell below has surface antigen CD45；
(vii) 0.3% cell below has surface antigen Actin；
(viii) 0.5% cell below has surface antigen CD14；
(ix) 0.1% cell below has surface antigen HLA-DR；
In a preferred embodiment of the present invention, mescenchymal stem cell after freezing have selected from the group below one or more
Feature:
(v) 1% cell below has surface antigen CD34；
(vi) 1% cell below has surface antigen CD45；
(vii) 0.3% cell below has surface antigen Actin；
(viii) 0.5% cell below has surface antigen CD14；
(ix) 0.1% cell below has surface antigen HLA-DR；
In a preferred embodiment of the present invention, mescenchymal stem cell after freezing have selected from the group below one or more
Feature:
(vi) 0.5% cell below has surface antigen CD45；
(vii) 0.5% cell below has surface antigen Actin；
(viii) 0.5% cell below has surface antigen CD14；
(ix) 0.1% cell below has surface antigen HLA-DR；
In a preferred embodiment of the present invention, mescenchymal stem cell after freezing have selected from the group below one or more
Feature:
(vii) 0.3% cell below has surface antigen Actin；
(viii) 0.5% cell below has surface antigen CD14；
(ix) 0.1% cell below has surface antigen HLA-DR.
In a preferred embodiment of the present invention, it is described freeze after mescenchymal stem cell do not have surface antigen CD34,
One of surface antigen CD45, surface antigen CD14, surface antigen HLA-DR or a variety of
The beneficial effects of the present invention are:
One, the present invention with freezing and storing umbilical mesenchymal stem cells and can make the navel after freezing using a kind of specific frozen stock solution
Band mescenchymal stem cell keeps activity；
Two, the present invention with freezing and storing umbilical mesenchymal stem cells and can make the navel after freezing using a kind of specific frozen stock solution
Band mescenchymal stem cell keeps differentiation stemness；
Three, present invention employs the volumes of formulation of optimization and concentration of component, frozen stock solution total volume 1-3mL has good
Freeze effect.
Detailed description of the invention
To describe the technical solutions in the embodiments of the present invention more clearly, make required in being described below to embodiment
Attached drawing is briefly described, it should be apparent that, drawings in the following description are only some embodiments of the invention, for
For those of ordinary skill in the art, without creative efforts, it can also be obtained according to these attached drawings other
Attached drawing, in which:
Fig. 1 is to freeze front and back umbilical cord mesenchymal stem cells Morphological comparison figure in embodiment one；
Fig. 2 is to freeze rear mescenchymal stem cell flow cytometer detection test map in embodiment two；
Fig. 3 be in embodiment three alcian blue dyeing detection umbilical cord mesenchymal stem cells freeze after can try hard at cartilage；
Fig. 4 is the osteogenic ability after umbilical cord mesenchymal stem cells freeze in example IV；
Fig. 5 is the umbilical cord mesenchymal stem cells after freezing in embodiment five into fatty ability.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiments of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's all other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition such as Sambrook et al.,
Molecular cloning: described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989)
Condition, or according to the normal condition proposed by manufacturer.
Embodiment one:
A kind of cryopreservation methods of umbilical cord mesenchymal stem cells, including step are provided are as follows:
(1) it washs umbilical cord tissue (removing haemocyte): the physiological saline of equivalent being added in the centrifuge tube of Xiang Hanyou umbilical cord, twists
Tight lid shakes 3min sufficiently to wash umbilical cord tissue, and then static 3-5min, separates different phases, suck lower layer's water phase；Weight
It operates more than multiple three times, until subnatant is more limpid.
(2) ultrasonic-microwave crushes: inhaling after abandoning physiological saline, adds the DMEM culture solution isometric with umbilical cord of preheating, set perseverance
In temperature microwave crusher, 37 DEG C of ultrasonic-microwaves are crushed.
(3) collect precipitating: digestion finishes, and 2000rpm is centrifuged 10min, discards the postdigestive umbilical cord in upper layer, collects two pipes
Bottom sediment adds DMEM culture solution to washed once to 50ml, 1000rpm centrifugation 8min to a new centrifuge tube.
(4) it filters and counts: adding DMEM culture solution to 50ml, mix, the filtering removal of 100um filter is the tissue of digestion
Block adds DMEM culture solution to 50ml, draws 1ml and count cell concentration and vigor.
(5) inoculated and cultured: 1000rpm centrifugation 8min washed once, according to the amount adjustment inoculum density inoculation for counting cell
To T75 culture bottle, wherein inoculum density are as follows: be inoculated with 12ml umbilical cord amount in general each T75 culture bottle, i.e. 50ml umbilical cord separates
The cell inoculation arrived sets 37 DEG C, 5%CO to 4 T75 culture bottles2Culture.
(6) it passes on: about 1-2 days adherent, a small amount of adherent mescenchymal stem cells of appearance in 3 days or so, 5-7 days patches of culture after inoculation
It when parietal cell is in colony, is passed on Trypsin EDTA solution digestion, 2ml, digestion time 1.5- is added in every T75 culture bottle
2.5min collects and counts cell, by 5*103/cm2(i.e. according to the ratio of primary adherent situation 1:1-2) passage is thin after passage
Intracellular growth becomes faster, and can pass on again within general three days, according to cell growth status, passes on according to the ratio of 1:2-3, ordinary circumstance
Under, P2 is decidedly superior to 2 × 10 for the number of umbilical cord mesenchymal stem cells7。
(7) it freezes: by 105-108Quickly with the resuspension of 1-3mL frozen stock solution, frozen stock solution contains the umbilical cord mesenchymal stem cells of/mL
Dimethyl sulphoxide solution that human serum albumin solution that mass fraction is 15%, mass fraction are 10%, 3ng/mL are intravascular
Skin cell growth factor, 2ng/mL fibrous bud Porcine HGF, the 3ng/mL interleukin 6 factor, 20ng/mL hepatocyte growth factor
Son, 15ng/mL transforming growth factor, wherein the mass ratio of human serum albumin solution and dimethyl sulphoxide solution is 2:1.It will be thin
It born of the same parents and freezes liquid mixture and is moved rapidly into cryopreservation tube, cryopreservation tube is placed in Slow-rate freezing instrument (Control Rate
Freezer), setting Slow-rate freezing instrument makes -1 DEG C/min of temperature fall off rate, until -80 DEG C, and at least protected in -80 DEG C
Liquid nitrogen container is moved into again after holding 24 hours.
(8) it recovers: the umbilical cord mesenchymal stem cells taking-up in cryopreservation tube being quickly placed into 37 DEG C of water baths, to frozen stock solution
Cell is moved into DMEM culture medium together with frozen stock solution after thawing.By 1 × 106Umbilical cord mesenchymal stem cells and 10uM DiD fluorescence contaminate
Material (Life Technologies&Molecular Probes, USA) is incubated for 50 minutes at 37 DEG C, and DiD fluorescent dye can be with
To detect cell viability in conjunction with living cells.Testing result is shown in Fig. 1, show to freeze the vigor of front and back umbilical cord mesenchymal stem cells without
Significant difference.
Embodiment two:
Freeze the identification of rear umbilical cord stem cells
It is resuspended after the P2 cultivated with embodiment one is centrifuged for umbilical cord stem cells；Cell concentration is adjusted after cell count
For l × 108/ L is anti-with mixture (Cocktail) monoclonal antibody room temperature of people anti-CD105, CD90, CD34 and CD45 respectively
30min is answered, cell is resuspended with PBS, is detected (Fig. 2) with flow cytometer.
Testing result is shown in Fig. 2 and carries out cell surface antigen to umbilical cord stem cells by flow cytometer it is found that freezing front and back
Label expression analysis shows umbilical cord mesenchymal stem cells after changing frozen stock solution and freezing phenotype without substantially changeing.
Embodiment three:
Rear umbilical cord stem cells are frozen into cartilage aptitude tests
Using the cell that embodiment one is frozen as of the present invention group, carry out into cartilage aptitude tests.In vitro to cartilage direction
After differentiation culture 3-4 week, see Fig. 3 it is found that alcian blue dyeing show the umbilical cord mesenchymal stem cells that are frozen with embodiment one and
The umbilical cord mesenchymal stem cells not the frozen ability having the same to cartilage differentiation in vitro, ability is without significant difference.
Example IV:
Freeze rear umbilical cord stem cells osteogenic ability test
Using the cell that embodiment one is frozen as of the present invention group, osteogenic ability test is carried out.In vitro to skeletonization direction point
After changing culture 3 weeks, Fig. 4 is seen it is found that alizarin red S dyeing is shown the umbilical cord mesenchymal stem cells frozen with embodiment one and do not frozen
The umbilical cord mesenchymal stem cells the deposited ability having the same to Osteoblast Differentiation in vitro, ability is without significant difference.
Embodiment five:
Rear umbilical cord stem cells are frozen into fatty aptitude tests
Using the cell that embodiment one is frozen as of the present invention group, carry out into fatty aptitude tests.In vitro at fat side
To after differentiation culture 2 weeks, oil red O stain shows the umbilical cord mesenchymal stem cells frozen with embodiment one and the umbilical cord that does not freeze
The mescenchymal stem cell ability having the same to Osteoblast Differentiation in vitro, ability is without significant difference.
Following is the definition of term as used in this specification.
As used herein, term " more than " and " following " include this number, such as " 95% or more " refer to >=95%, " 0.2% with
Under " refer to≤0.2%.
Human serum albumins
Human serum albumins (Human Serum Albumin, abbreviation HSA) is the protein in human plasma.Human seralbumin
Albumen is widely used as Plasma volume expansion agent.The human serum albumins used in the present invention is the pure egg of clinical grade human serum
It is white, it can directly apply to human body.
Dimethyl sulfoxide
Dimethyl sulfoxide (DMSO) is a kind of organic compounds containing sulfur, is the transparency liquid of no color or smell under room temperature.DMSO
It is also a kind of permeability protective agent, can reduce cell freezing point, reduce the formation of ice crystal, mitigates free radical to cell damage, change
Become biomembrane to the permeability of electrolyte, drug, poisonous substance and metabolite.
Trophic factors
Trophic factors is generated by various kinds of cell, have extensively adjust cell function effect peptide molecule, cell because
Son acts not only on immune system and hemopoietic system, go back wide application in nerve, endocrine system, to cell-cell interaction,
The Proliferation, Differentiation and effector function of cell have important adjustment effect.The cell factor used in the present invention is clinical grade cell
The factor can directly apply to human body.
Umbilical cord
Umbilical cord is to link structure between fetus and placenta.Shape such as rope, surface glossy clear, include connective tissue and
One umbilical vein, a pair of of arteria umbilicalis.Umbilical vein passes through the blood sinus of liver, arteria umbilicalis and fetus active pulse-phase along fetus stomach wall inner face
Lead to.The other end of both blood vessels forms many capillary networks connected each other, is distributed in placental villi.Pass through tire
The osmosis of disk chorioepithelium carries out mass exchange between maternal blood in fetus disk liquid and intervillous space.In the present invention,
Umbilical cord tissue or umbilical cord raw material are derived from the umbilical cord tissue of healthy newborn.
The mescenchymal stem cell in umbilical cord source
As used herein, term " mescenchymal stem cell in umbilical cord source ", " huMSCs " or " Human Umbilical
Cord tissue-derived mesenchymal progenitor cells " meaning is identical, may be used interchangeably.The present invention
The mescenchymal stem cell in the umbilical cord source of the excellent human origin of the mescenchymal stem cell in the umbilical cord source used.
The separation and culture that conventional method carries out huMSCs, including step can be used in those skilled in the art
Are as follows:
A) it washs umbilical cord tissue (removing haemocyte): physiological saline is added, into umbilical cord tissue sufficiently to wash umbilical cord group
It knits, separates different phases, suck lower layer's water phase；It repeats above operation until subnatant is more limpid.
B) ultrasonic-microwave crushes: crushing to umbilical cord tissue.
C) collect precipitating: discarding the smashed umbilical cord block in upper layer, collect bottom sediment to a new centrifuge tube, add DMEM with
Centrifuge washing.
D) it filters and counts: adding DMEM, mix, filtering removes indigested tissue block, and DMEM, absorption 1ml is added to count thin
Born of the same parents' amount and vigor.
E) inoculated and cultured: centrifuge washing is primary, is seeded to T75 culture bottle according to the amount adjustment inoculum density for counting cell
(inoculum density: it is inoculated with 12ml umbilical cord amount in general each T75 culture bottle, i.e., the isolated cell inoculation of 50ml umbilical cord is to 4
T75 culture bottle), it is cultivated.
F) pass on: after inoculation about 1-2 days it is adherent, there is within 3 days or so a small amount of adherent mescenchymal stem cell, culture to it is adherent carefully
It when born of the same parents are in colony, is passed on Trypsin EDTA solution digestion, 2ml, digestion time 1.5- is added in every T75 culture bottle
2.5min collects and counts cell, passes on according to the ratio of primary adherent situation 1:1-2, and cell growth becomes faster after passage, and one
As can pass on again within three days, according to cell growth status, passed on according to the ratio of 1:2-3, under normal circumstances, P2 is between umbilical cord
The number of mesenchymal stem cells is decidedly superior to 2 × 107, can satisfy cell concentration needed for general treatment.
The antigen of the mescenchymal stem cell in umbilical cord source detects
The mescenchymal stem cell in the umbilical cord source that the present invention uses has high purity and activity.
Those skilled in the art can be used conventional method and detect to mescenchymal stem cell surface antigen, such as
Overflow-type cytometry etc..
The mescenchymal stem cell in umbilical cord source have a variety of specific antigens and receptor, mainly have: CD29, CD73, CD90,
CD105 etc..
Ratio >=92% of the mescenchymal stem cell with CD29 antigen in total mescenchymal stem cell, more preferably >=
95%, more preferably >=98%.
Ratio >=85% of the mescenchymal stem cell with CD73 antigen in total mescenchymal stem cell, more preferably >=
90%, more preferably >=92%.
Ratio >=92% of the mescenchymal stem cell with CD90 antigen in total mescenchymal stem cell, more preferably >=
95%, more preferably >=96%.
Ratio >=85% of the mescenchymal stem cell with CD49d antigen in total mescenchymal stem cell, more preferably >=
90%, more preferably >=95%.
The negative indication of the mescenchymal stem cell in umbilical cord source includes: CD34, CD45 etc..
Ratio≤2% of the mescenchymal stem cell with CD34 in total mescenchymal stem cell, more preferably≤1%, more preferably
Ground≤0.5%, most preferably without CD34.
Ratio≤2% of the mescenchymal stem cell with CD45 in total mescenchymal stem cell, more preferably≤1%, more preferably
Ground≤0.5%, most preferably without CD45.
Those skilled in the art can be used conventional method to huMSCs carry out using, processing, application etc. operation.
Such as: before every batch of huMSCs granting or use, it is necessary to pass through sterile, endotoxin and mycoplasma inspection and DNA is the same.
The cell of every batch of granting will meet cell viability>=95%, cell purity (positive indication>=95%, negative indication<2%).
The anxious poison of huMSCs, allergy testing result are negative, and have a corresponding examining report.
Frozen stock solution composition provided by the invention, it contains a effective amount of human serum albumins and dimethyl sulfoxide, statement
The cell factor and pharmaceutically acceptable carrier of concentration.The pharmaceutically acceptable carrier is compound electrolyte injection
Liquid, the Multiple electrolytes injection is commercially available to be bought, and contains chlorine in the general every 1000ml of Multiple electrolytes injection
Change sodium 5.26g, sodium gluconate 5.02g, Sodium acetate trihydrate 3.68g, potassium chloride 0.37g, magnesium chloride hexahydrate 0.30g.
In general, mescenchymal stem cell can be formulated in nontoxic, inert and pharmaceutically acceptable aqueous carrier medium
In, in physiological saline, wherein pH is usually about 5-8, preferably, pH is about 7-8.
As used herein, term " effective quantity " or " effective dose " refer to can to umbilical cord mesenchymal stem cells generate function or
Active amount.
As used herein, the ingredient of " pharmaceutically acceptable " is suitable for people and/or mammal and without excessively bad
Side reaction (such as toxicity, stimulation and allergy), i.e., with the substance of reasonable benefit/risk ratio.Term " can pharmaceutically connect
The carrier received " refers to the carrier for Therapeutic Administration, including various excipient and diluent.
The carrier that pharmaceutical composition of the invention contains includes (but being not limited to): salt water, buffer, glucose, water, sweet
Oil, ethyl alcohol, and combinations thereof.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair
Equivalent structure or equivalent flow shift made by bright description is applied directly or indirectly in other relevant technology necks
Domain is included within the scope of the present invention.
Claims (10)
1. a kind of umbilical cord mesenchymal stem cells frozen stock solution, which is characterized in that molten including human serum albumin solution, dimethyl sulfoxide
Liquid, trophic factors and pharmaceutically acceptable carrier, the trophic factors are blood vessel endothelial cell growth factor VEGF, conversion life
One of long factor TGF, interleukin 6 IL-6, fibrous bud Porcine HGF bFGF, hepatocyte growth factor HGF or more
Kind.
2. umbilical cord mesenchymal stem cells frozen stock solution according to claim 1, which is characterized in that the human serum albumins is molten
Liquid is the human serum albumin solution that mass fraction is 5-15%；The dimethyl sulphoxide solution is two that mass fraction is 5-10%
Methyl sulfoxide solution；The concentration of the vascular endothelial growth factor is 2-3ng/mL, the fibrous bud Porcine HGF
Concentration be 2-6ng/mL, the concentration of the interleukin 6 is 2-3ng/mL, and the concentration of the hepatocyte growth factor is 15-
20ng/mL, the concentration of the transforming growth factor are 10-15ng/mL；The human serum albumin solution and the dimethyl are sub-
The mass ratio of sulfolane solution is 1-2:1；The pharmaceutically acceptable carrier is Multiple electrolytes injection；The umbilical cord mesenchyma
Stem cell cryopreserving liquid is unit dosage form, and the volume of the unit dosage form is≤4mL.
3. umbilical cord mesenchymal stem cells frozen stock solution according to claim 2, which is characterized in that the human serum albumins is molten
Liquid is the human serum albumin solution that mass fraction is 8-12%；The dimethyl sulphoxide solution is two that mass fraction is 7-9%
Methyl sulfoxide solution；The mass ratio of the human serum albumin solution and the dimethyl sulphoxide solution is 1-1.5:1；The list
The volume of first dosage form is≤2mL.
4. a kind of purposes of umbilical cord mesenchymal stem cells frozen stock solution, which is characterized in that the umbilical cord mesenchymal stem cells frozen stock solution
Application in umbilical cord mesenchymal stem cells freeze.
5. the purposes of umbilical cord mesenchymal stem cells frozen stock solution according to claim 4, which is characterized in that every 105-108/mL
The umbilical cord mesenchymal stem cells apply the umbilical cord mesenchymal stem cells frozen stock solution of each unit.
6. the purposes of umbilical cord mesenchymal stem cells frozen stock solution according to claim 5, which is characterized in that every 106-107/mL
The umbilical cord mesenchymal stem cells apply the umbilical cord mesenchymal stem cells frozen stock solution of each unit.
7. the purposes of umbilical cord mesenchymal stem cells frozen stock solution according to claim 4, which is characterized in that the cryopreservation methods
- 80 DEG C are at the uniform velocity cooled to -1 DEG C/min of rate for the umbilical cord mesenchymal stem cells, is subsequently placed in liquid nitrogen container and protects for a long time
It deposits.
8. the purposes of umbilical cord mesenchymal stem cells frozen stock solution according to claim 4, which is characterized in that the umbilical cord after freezing
Mescenchymal stem cell has selected from the group below any or various features:
(i) 92% or more cell has surface antigen CD90；
(ii) 85% or more cell has surface antigen CD73；
(iii) 92% or more cell has surface antigen CD29；
(iv) 85% or more cell has surface antigen CD49d；
Preferably, the umbilical cord mesenchymal stem cells after freezing have selected from the group below any or various features:
(i) 95% or more cell has surface antigen CD90；
(ii) 90% or more cell has surface antigen CD73；
(iii) 95% or more cell has surface antigen CD29；
(iv) 90% or more cell has surface antigen CD49d；
It is highly preferred that the umbilical cord mesenchymal stem cells after freezing have selected from the group below any or various features:
(i) 96% or more cell has surface antigen CD90；
(ii) 92% or more cell has surface antigen CD73；
(iii) 98% or more cell has surface antigen CD29；
(iv) 95% or more cell has surface antigen CD49d.
9. the purposes of umbilical cord mesenchymal stem cells frozen stock solution according to claim 4, which is characterized in that filled between after freezing
Matter stem cell has selected from the group below any or various features:
(v) 2% cell below has surface antigen CD34；
(vi) 2% cell below has surface antigen CD45；
(vii) 0.3% cell below has surface antigen Actin；
(viii) 0.5% cell below has surface antigen CD14；
(ix) 0.1% cell below has surface antigen HLA-DR；
Preferably, the mescenchymal stem cell after freezing has selected from the group below any or various features:
(v) 1% cell below has surface antigen CD34；
(vi) 1% cell below has surface antigen CD45；
(vii) 0.3% cell below has surface antigen Actin；
(viii) 0.5% cell below has surface antigen CD14；
(ix) 0.1% cell below has surface antigen HLA-DR；
It is highly preferred that the mescenchymal stem cell after freezing has selected from the group below any or various features:
(vi) 0.5% cell below has surface antigen CD45；
(vii) 0.5% cell below has surface antigen Actin；
(viii) 0.5% cell below has surface antigen CD14；
(ix) 0.1% cell below has surface antigen HLA-DR；
Most preferably, the mescenchymal stem cell after freezing has selected from the group below any or various features:
(vii) 0.3% cell below has surface antigen Actin；
(viii) 0.5% cell below has surface antigen CD14；
(ix) 0.1% cell below has surface antigen HLA-DR.
10. the purposes of umbilical cord mesenchymal stem cells frozen stock solution according to claim 4, which is characterized in that it is described freeze after
Mescenchymal stem cell do not have surface antigen CD34, surface antigen CD45, surface antigen CD14, in surface antigen HLA-DR
It is one or more.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910451040.0A CN110199984A (en) | 2019-05-28 | 2019-05-28 | A kind of umbilical cord mesenchymal stem cells frozen stock solution and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910451040.0A CN110199984A (en) | 2019-05-28 | 2019-05-28 | A kind of umbilical cord mesenchymal stem cells frozen stock solution and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110199984A true CN110199984A (en) | 2019-09-06 |
Family
ID=67789032
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910451040.0A Pending CN110199984A (en) | 2019-05-28 | 2019-05-28 | A kind of umbilical cord mesenchymal stem cells frozen stock solution and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110199984A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111418580A (en) * | 2020-05-25 | 2020-07-17 | 山东万能干细胞生物技术有限公司 | Stem cell cryopreservation solution and cryopreservation method |
| CN113133444A (en) * | 2021-03-31 | 2021-07-20 | 北京益华生物科技有限公司 | Umbilical cord tissue cryopreservation liquid and preparation method thereof, umbilical cord tissue cryopreservation method and culture method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106982821A (en) * | 2017-05-22 | 2017-07-28 | 安徽瑞杰赛尔生物科技有限公司 | Umbilical cord mesenchymal stem cells clinic freezes protection liquid composition and application thereof |
| CN107810948A (en) * | 2017-10-04 | 2018-03-20 | 刘劼 | A kind of serum-free frozen stock solution of human umbilical cord mesenchymal stem cells |
| WO2018199482A1 (en) * | 2017-04-26 | 2018-11-01 | Jang Ho Lee | Composition for cryopreservation of cells or tissues including mesenchymal stem cell culture medium |
| CN108812645A (en) * | 2018-07-20 | 2018-11-16 | 吉林济惠生物科技有限公司 | A kind of cells frozen storing liquid of human umbilical cord mesenchymal stem cells |
-
2019
- 2019-05-28 CN CN201910451040.0A patent/CN110199984A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018199482A1 (en) * | 2017-04-26 | 2018-11-01 | Jang Ho Lee | Composition for cryopreservation of cells or tissues including mesenchymal stem cell culture medium |
| CN106982821A (en) * | 2017-05-22 | 2017-07-28 | 安徽瑞杰赛尔生物科技有限公司 | Umbilical cord mesenchymal stem cells clinic freezes protection liquid composition and application thereof |
| CN107810948A (en) * | 2017-10-04 | 2018-03-20 | 刘劼 | A kind of serum-free frozen stock solution of human umbilical cord mesenchymal stem cells |
| CN108812645A (en) * | 2018-07-20 | 2018-11-16 | 吉林济惠生物科技有限公司 | A kind of cells frozen storing liquid of human umbilical cord mesenchymal stem cells |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111418580A (en) * | 2020-05-25 | 2020-07-17 | 山东万能干细胞生物技术有限公司 | Stem cell cryopreservation solution and cryopreservation method |
| CN113133444A (en) * | 2021-03-31 | 2021-07-20 | 北京益华生物科技有限公司 | Umbilical cord tissue cryopreservation liquid and preparation method thereof, umbilical cord tissue cryopreservation method and culture method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5341059B2 (en) | Stem cell suspension | |
| US20080118477A1 (en) | Umbilical cord mesenchymal stem cells support cord blood hematopoiesis | |
| WO2011101834A1 (en) | A method for obtaining mesenchymal stem cells, media, methods and composition thereof | |
| EP2574665A1 (en) | Procurement, isolation and cryopreservation of endometrial/menstrual cells | |
| EP3015545A1 (en) | Trehalose and dextran-containing solution for transplanting mammalian cells | |
| EP1999250B1 (en) | Method of cultivation of human mesenchymal stem cells, particularly for the treatment of non-healing fractures, and bioreactor for carrying out this cultivation method | |
| KR20100087705A (en) | Optimised and defined method for isolation and preservation of precursor cells from human umbilical cord | |
| WO2009103818A1 (en) | Methods for obtaining progenitor cells and uses thereof in the treatment of tissue or organ damage | |
| US20230227786A1 (en) | PREPARATION METHOD AND RECOVERY METHOD OF PARIDUVAL MESENCHYMAL STEM CELLS (PMSCs) | |
| US20220340869A1 (en) | Cell population comprising mesenchymal stem cells derived from fetal appendage, method for producing the same, and pharmaceutical composition | |
| JP2022084665A (en) | Stem cell therapy based on adipose-derived stem cells | |
| KR101178424B1 (en) | Process of In Vitro Mass Production of Clinical-Grade Red Blood Cells | |
| JP2018531269A6 (en) | Stem cell therapy based on adipose-derived stem cells | |
| CN110199984A (en) | A kind of umbilical cord mesenchymal stem cells frozen stock solution and application thereof | |
| KR20100084620A (en) | Cell composition for tissue regeneration | |
| JP2023103416A (en) | Therapeutic agent for dilated cardiomyopathy | |
| CA3168330A1 (en) | Method for treating chronic graft versus host disease | |
| CN105695401B (en) | The preparation of all stem cells of a kind of umbilical artery and vein blood vessel and store method | |
| CN112250798B (en) | Preparation method of hydrogel and mesenchymal stem cells | |
| JP7140752B2 (en) | Cell population containing fetal appendage-derived adhesive cells, method for producing the same, and pharmaceutical composition | |
| CN117701500B (en) | Culture method and application of mesenchymal stem cells | |
| CN110840914B (en) | Method for alleviating or improving vascular disorders using cell therapeutic agent | |
| Ghoneim et al. | Isolation of Bone Marrow and Adipose-Derived Mesenchymal Stromal Cells | |
| KR20230017722A (en) | Composition for wound healing or accelerating wound healing comprising exosomes derived from cord blood progenitor cell | |
| CN112841176A (en) | Long-term cryopreservation medium for adipose-derived stem cells and preparation method and application thereof |
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: 20190906 |