CN109090100A - A kind of mesenchymal stem cell cryopreserving liquid and preparation method thereof and application method - Google Patents
A kind of mesenchymal stem cell cryopreserving liquid and preparation method thereof and application method Download PDFInfo
- Publication number
- CN109090100A CN109090100A CN201810982226.4A CN201810982226A CN109090100A CN 109090100 A CN109090100 A CN 109090100A CN 201810982226 A CN201810982226 A CN 201810982226A CN 109090100 A CN109090100 A CN 109090100A
- Authority
- CN
- China
- Prior art keywords
- stem cell
- cell
- mesenchymal stem
- cryopreserving liquid
- serum
- 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
- 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
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)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a kind of mesenchymal stem cell cryopreserving liquid and preparation method thereof and application method, the mesenchymal stem cell cryopreserving liquid includes Rock inhibitor Y27632, dimethyl sulfoxide, human serum albumin, hydroxyethyl starch etc..Mesenchymal stem cell cryopreserving liquid of the invention is suitable for slow cryopreservation method, not only easy to operate, but also the formula also has the advantages that low toxicity, economic cost are low, can also maintain the activity of mescenchymal stem cell for a long time, freezes and works well and more safe and reliable.
Description
Technical field
The invention belongs to cell technology fields, and in particular to a kind of mesenchymal stem cell cryopreserving liquid and preparation method thereof with make
Use method.
Background technique
Mescenchymal stem cell (mesenchymal stem cells, MSC) be a group have height self-renewal capacity and
The adult stem cell of differentiation potential receives much attention due to having many advantages, such as that immunoregulation, secrete cytokines, materials facilitate, at
For the ideal seed cell of cell therapy tissue damage reparation.According to U.S. Present clinical experimental data base Clinical Trials
Data show that clinical test relevant to mescenchymal stem cell has been directed to more than 300 kinds of diseases at present, be singly the U.S. carry out with
The relevant clinical test of mescenchymal stem cell has just reached 650 more than one piece, and main disease includes bone injury, nervus retrogression disease
Disease, diabetes, myocardial ischemia, myasthenia gravis, hepatic injury etc..As it can be seen that mescenchymal stem cell research is in cell therapy, group weaver
The fields such as journey have particularly important application value, are that clinical application research is most after candidate stem cell, and most mature
A kind of adult stem cell.
Since mescenchymal stem cell still has the latent of Multidirectional Differentiation after continuous passage culture and cryopreservation resuscitation in vitro
Can, it can be used as ideal seed cell applied to injuries of tissues and organs reparation caused by aging and lesion.But different tissues come
MSCs (such as marrow, umbilical cord, the Cord blood and placenta) proportion in source has very big difference, from 0.001% to 0.01% etc.,
Therefore ideal cell seed is limited.In addition, the transplantation treatment of mescenchymal stem cell, needs to cultivate in vitro and be expanded to a fixed number
It could be applied after amount, but transition amplification will lead to cell ageing and function is lost.Therefore, how by useful, " mesenchyma is dry thin
Born of the same parents' seed " stores, in addition amplification it is enough stored to establish stem cell resources bank, be guarantee mescenchymal stem cell treatment
The premise of research and application, so developing effective one kind, energy long-term preservation mescenchymal stem cell and remaining original multidirectional
The Cryopreservation Technology of differentiation capability is particularly important.
There are mainly two types of existing most cells Cryopreservation Technologies: slow cryopreservation method (i.e. programmed cooling method) and vitrifying
Method, wherein the method for slow cryopreservation uses relatively broad.Slow cryopreservation technology is the cryoprotection that cell is first placed on to low concentration
It is pre-processed in agent, then above-mentioned cell is cooled down together with solution with slower rate with programmed cooling instrument or low temperature refrigerator；
Moisture icing in Extracellular solution increases solution concentration, and moisture intracellular is oozed out outward by cell membrane, and cell volume is shunk,
Solution concentration intracellular increases, and with the decline of temperature, the above process continues, when cooling to certain low temperature, then fast prompt drop
Temperature is to liquid nitrogen temperature, and long-term preservation is at this temperature.It can be seen that the selection of freezing protective agent is particularly critical.
The agent that freezes common at present has three categories: permeability protective agent, semipermeability protective agent and high molecular polymer, it
Protection mechanism it is as follows:
(1) permeability protective agent
Permeability cryoprotector can be entered into the cell by cell membrane, and common permeability cryoprotector is main
There are glycerol, dimethyl alum, propylene glycol to kowtow, ethylene glycol etc..Such cryoprotector belongs to low molecule neutral substance more, how readily soluble
Yu Shui, strong with water molecules ability, easy penetrating cell film enters cell interior, reduces the freezing point of cell in the cell in solution
In easily bound water molecule occur aquation, make solution viscosity increase, to weaken the crystallization process of water, reach protection
Purpose.Extracellular water can be promoted to enter cell when rewarming, alleviate damage caused by permeability swelling and dilute in non-frozen solution
The concentration of electrolyte reduces solute damage.
Protectant selection will be depending on the type for being saved cell, and DMSO (dimethyl sulfoxide) is mainly used for tissue or thin
The preservation of born of the same parents' suspension, ethylene glycol (EG) and 1,2-PD are chiefly used in various ovums and the deep-bed drying of embryo, and glycerol is main
Preservation for sperm.Wherein, DMSO is generally acknowledged widely applied protective agent.DMSO energy penetrating cell film enters cell,
Stable hydrone and intracellular protein intracellular and by stablizing plasma membrane with the electrostatic interaction of phospholipid bilayer, alleviate slow
The difference of intraor extracellular osmotic pressure caused by being frozen during jelly due to extracellular hydrone.Meanwhile DMSO can also be with hydrone shape
At hydrogen bonding compound, hydrone is held, mitigates the redistribution of hydrone in freezing process.Mitigate in sample cooling intracellular
Hydrone outflow mitigates extracellular water in sample rewarming to intracellular stream, to protect cell.Although but in low temperature
Under can be with stabilizing cell membrane albumen, but albumen can be made to be denaturalized as the temperature rises, to generate toxicity to cell.
(2) semipermeability protective agent
Semipermeability protective agent cannot pass through cell membrane, but can penetrating cell wall, mainly have monosaccharide, disaccharides, amino acid
With low molecular weight polycaprolactone zoarium etc..This substance is usually small molecule carbohydrate, can be dissolved in water, but not can enter cell, it makes cell exist
It is first partially dehydrated before freezing, it is concentrated between cell wall and cell membrane, forms buffer layer, prevent the generation of ice crystal, mechanically
Protect cell membrane.
(3) high molecular polymer
Common high molecular polymer includes that polyethylene adjoins pyrrolidone (PVP), polyethylene glycol (PEG), albumin
(Albumin), hydroxyethyl starch (HES), glucan (Dextran), ficoll (Ficoll) etc..This substance is usually big point
Sub- substance, not can enter cell, can preferentially combine with the hydrone in solution, reduces the content of Free water in solution, makes ice
Point reduces, and reduces the formation of ice crystal simultaneously as its molecular weight is big, reduces the electrolyte concentration in solution, to mitigate molten
Matter is damaged and impermeability high molecular polymer can be subtracted by reducing the intracellular required protectant concentration of permeability
The whole toxicity of light cryoprotector, moreover it is possible to promote solution vitrifying.In freezing, such protective agent is mainly protected with permeability
Agent is used in combination, and when cell being promoted to complete dehydration rewarming, provides a hypertonic environment, prevents moisture from entering cell and causes fastly very much
Cell expansion destroys.For example, the PEG of macromolecule can form glutinous layer in cell surface, increase the viscosity of cell peripheral solution,
Inhibit the growth of ice crystal or form crystallite, to protect cells from the damage of ice crystal.Hydroxyethyl starch (HES) not only protect by low temperature
It is good to protect effect, and to human toxicity very little, adds it in preservation liquid and freezes marrow, after rewarming not without elution or elution
Only, it inputs in experimental animal body and is reacted without overt toxicity.
Since single protective agent effect is limited, usually protective agent is used in combination.Until currently, freezing protectant research
It is still the research hotspot of low temperature preservation field.Therefore, the characteristics of how freezing agent according to difference, developing can be with long-term preservation
Hypotoxicity even nontoxic mesenchymal stem cell cryopreserving liquid be of great significance to the security application of stem cell.
The frozen stock solution of mescenchymal stem cell is formed by two kinds or more of protective agent compatibilities at present, wherein with DMSO and
What fetal calf serum (fetal bovine serum, FBS) or human serum albumins (Human albumin) formed freezes agent most
It is common.
If any scholar using it is general freeze formula of liquid (10%DMSO+40%FBS+50%DMEM/F12) inquire into its
When the effect of human adipose mesenchymal stem cells cryopreservation resuscitation, as a result, it has been found that as the time frozen extends, MSCs cell recovery
Motility rate is declined slightly, but this scheme freeze 6 months after fat mesenchymal dry carefully averagely motility rate is more than 85%, MSCs cell
The biological characteristics such as form, surface marker, proliferation, form and induction ability are compared with cell when not freezing, equal nothing
Significant difference.
And 10uM is added in common 10%DMSO+20%FBS+70%DMEM frozen stock solution in the team such as Gauthaman K.
Rock inhibitor Y-27632 (a kind of anti-apoptotic inhibitor) when, after freezing 3 months recover discovery, contain anti-apoptotic inhibitor
ROCK inhibitor Y-27632 is remarkably improved the motility rate of cell recovery or even promotes to be proliferated, and karyotyping also found this
Combined method does not influence the biological heredity characteristic of MSCs, is a kind of safely and effectively cryopreservation methods.
But above two frozen stock solution ingredient be unable to do without DMSO, serum product and remaining incomplete culture medium ingredient.
DMSO in these ingredients easily causes a system such as pernicious patient, headache, abdominal cramps because having potential Side effect
Column adverse reaction.And serum, especially animal blood serum, there may be the risk for carrying transmitted virus (such as prion or some to cause
The protein of immune response), along with the cell of the component of animal blood serum, source and production batch to culture might have it is bad
Or inconsistent influence.Therefore, how to reduce DMSO using concentration and develop alternative serum frozen stock solution it is particularly important.
In order to reduce DMSO using concentration and the product for developing alternative serum, we have found Ji by searching document
The team such as Min Seo are in the influence of the frozen stock solution for the studying various concentration DMSO mescenchymal stem cell amnion-derived to people, hair
Now either common 10%DMSO+30%FBS freezes formula or 5% and 2.5% difference of DMSO composition freezes agent and matches
Side has amnion-derived mescenchymal stem cell and freezes protecting effect, wherein with 5% (v/v) DMSO+60m mol/L
Trehalose+100 (ug/ml) Catalase+30uM zVAD-fmk effect is maximally efficient, also there is substitution to contain serum
Frozen stock solution.Although this effect is not grown, it is contemplated that trehalose (trehalose), Catalase (catalase) and
This combination of caspase inhibitor zvad-fmk is not only complicated for operation, but also various enzymes are expensive, therefore are difficult in the application
It promotes.
In addition, in terms of patent application, we it also seen that, application No. is 201310568189.X, denominations of invention are as follows: a kind of
Mesenchymal stem cell cryopreserving liquid and injection.It finds and common frozen stock solution (i.e. 10%DMSO+90%DMEM/F12, control
Group) it compares, novel frozen stock solution percent composition range exists: Bomaili A liquid 25~75%, 18AA5% Amino Acid Compound Injection
When 10~35%, 20% human serum albumin 1~50%, 5~20% DMSO, the novel mesenchymal stem cell cryopreserving of the invention is equal
89.2% or more.When Bomaili A liquid 40%, 18AA5% Amino Acid Compound Injection 25%, 20% human serum albumin 25%,
When DMSO 10%, such cryopreservation motility rate is up to 95.4%, is significantly higher than 92.2% [9] of Normal group.Though
Right but this scheme freezes 1 year and presents excellent effect, it is contemplated that its ingredient such as Bomaili A and amino acid injection
This compound electrolyte of liquid can have not yet to see so far report and verifying as the principle for freezing agent.
Another comes from number of patent application are as follows: and 200910167171.2, denomination of invention are as follows: stem cell cryopreserving liquid and stem cell
Cryopreservation methods.They are also illustrated that: using the people's AB serum of HES+10~80% of 5~10% volume DMSO and 3~6% weight at that time
Frozen stock solution when, freeze density in 1x106~4x 107When the umbilical cord mesenchymal stem cells of/ml, freezes and recover after a week, cell
Motility rate is 90% or more.Although this scheme that freezes only is suitable for short-term freezer storage, long-term jelly using more popularizing
It is still less desirable to deposit effect.
Summary of the invention
It is of the existing technology in order to solve the problems, such as, it is used for most of frozen stock solution DMSO in the prior art and serum dense
Height is spent, ingredient is relative complex, the undesirable these problems of Long-term Cryopreservation effect, and the present invention provides a kind of mescenchymal stem cell jellies
Liquid storage and preparation method thereof and application method, mesenchymal stem cell cryopreserving liquid of the invention have relatively simply, answer suitable for promoting
The features such as with, hypotoxicity, and mescenchymal stem cell being stored for a long time, while maintaining its biological characteristics.
The object of the present invention is to provide a kind of mesenchymal stem cell cryopreserving liquids.
The mesenchymal stem cell cryopreserving liquid of specific embodiment according to the present invention, the mesenchymal stem cell cryopreserving liquid packet
Include Rock inhibitor Y27632, dimethyl sulfoxide, human serum albumin, hydroxyethyl starch and serum-free stem cell media.
The mesenchymal stem cell cryopreserving liquid of specific embodiment according to the present invention, wherein the mescenchymal stem cell freezes
It is 5-10%, human serum albumin 5-10%, serum-free stem cell media that liquid storage, which includes dimethyl sulfoxide by percent by volume,
For 70-90%, Rock inhibitor Y27632 is 5-10umol/L and hydroxyethyl starch is 3-6%.
The mesenchymal stem cell cryopreserving liquid of specific embodiment according to the present invention, wherein the mescenchymal stem cell freezes
It is 5% that liquid storage, which includes dimethyl sulfoxide by percent by volume, human serum albumin 10%, and serum-free stem cell media is
75%, Rock inhibitor Y27632 are 7.5umol/L and hydroxyethyl starch is 6%.
The mesenchymal stem cell cryopreserving liquid of specific embodiment according to the present invention, wherein the serum-free stem cell training
Supporting base is DMEM/F-12 culture medium.
It is a further object of the present invention to provide the preparation methods of above-mentioned mesenchymal stem cell cryopreserving liquid.
The preparation method of the mesenchymal stem cell cryopreserving liquid of specific embodiment according to the present invention, the method includes with
Lower step: according to the frozen stock solution volume of cell density and quasi- configuration, hydroxyethyl starch, the Rock inhibitor of corresponding proportion are first taken
Y27632 and serum-free stem cell media, are gently suspended；Then the human serum albumins for adding corresponding ratio, is gently mixed
It is outstanding；Dimethyl sulfoxide is finally added dropwise, mixes, the as described mesenchymal stem cell cryopreserving liquid.
The preparation method of the mesenchymal stem cell cryopreserving liquid of specific embodiment according to the present invention, wherein the diformazan
The volume ratio 1:2:7 of base sulfoxide, the human serum albumin and the serum-free stem cell media.
The preparation method of the mesenchymal stem cell cryopreserving liquid of specific embodiment according to the present invention, wherein step (1)
In, the dimethyl sulfoxide is put into 4 DEG C of refrigerators before dropwise addition and 10-30min is pre-chilled, and is pre-chilled to 4 DEG C.
Another object of the present invention is to provide the application method of above-mentioned mesenchymal stem cell cryopreserving liquid.
The application method of the mesenchymal stem cell cryopreserving liquid of specific embodiment according to the present invention, the application method packet
Include following steps:
(1): first collecting cell to be frozen, digest, supernatant is abandoned in centrifugation；
(2): according to the cell density in frozen stock solution, calculating freezes frozen stock solution volume required for sample, at step (1)
Hydroxyethyl starch, Rock inhibitor Y27632 and serum-free stem cell media are added in cell to be frozen after reason, gently
It is suspended；Then human serum albumins is added, is gently suspended；Dimethyl sulfoxide is finally added dropwise, is freezed after mixing.
When operation, cell to be frozen is collected, is digested, supernatant is abandoned in centrifugation；Then into treated cell to be frozen
Sterile PBS is added, cell is counted, is centrifuged again after counting；According to freezing density (2-10 × 106A/ml) it calculates and intends
The total volume of freeze-stored cell, according to the overall accumulated amount frozen, be first added hydroxyethyl starch (HES) and Rock inhibitor Y27632,
Serum-free stem cell media, is gently suspended；Then human serum albumins (HSE) is being added, is gently being suspended；Finally it is added dropwise
DMSO is finally mixed, and freezing is transferred in liquid nitrogen container and saved.Wherein, DMSO must be added finally, and otherwise cell can quilt
The DMSO toxicity of high concentration is lethal.
The application method of the mesenchymal stem cell cryopreserving liquid of specific embodiment according to the present invention, wherein step (2)
In, cell density in frozen stock solution to 2-10 × 106A/ml.
The application method of the mesenchymal stem cell cryopreserving liquid of specific embodiment according to the present invention, in step (2), institute
State freezing specifically: frozen stock solution containing cell is dispensed into cryopreservation tube, then cryopreservation tube is put into program temperature reduction box, then
Program temperature reduction box is put into -80 DEG C of refrigerators 6 hours or more, transfers in liquid nitrogen container and saves.It can be marked on cryopreservation tube thin
Born of the same parents' title, date and quantity etc..
The recovery of MSCs cell:
Frozen stock solution containing MSCs cell is taken out from liquid nitrogen container, is quickly transferred to place 2min in 37 DEG C of water-baths,
Almost melt to ice, be transferred in the centrifuge tube containing 9ml PBS and wash, 200g is centrifuged 5 minutes, finally abandons supernatant, is added
After stem cell complete medium, it is inoculated into culture dish and cultivates.
Dimethyl sulfoxide (DMSO) permeability of cell membrane with higher in the present invention, is a kind of widely applied infiltration
Property cryoprotector, it can penetrating cell film enter cell, stable hydrone intracellular and intracellular protein and by with phosphatide
The electrostatic interaction of bilayer stablizes plasma membrane, alleviates intracellular caused by being frozen during slow freeze due to extracellular hydrone
The difference of exosmosis pressure.Simultaneously, moreover it is possible to form hydrogen bonding compound with hydrone, hold hydrone, mitigate moisture in freezing process
The redistribution of son.Sample cooling when mitigate intracellular water outflow, in sample rewarming mitigate extracellular water to
Intracellular stream, to protect cell.
And human serum albumins (HSE) and hydroxyethyl starch (HES) this substance are usually macromolecular substances, not can enter
Cell can be combined preferentially with the hydrone in solution, reduced the content of Free water in solution, reduced freezing point, and ice crystal is reduced
Formation simultaneously as its molecular weight is big, reduce the electrolyte concentration in solution, to mitigate solute damage and impermeable
Property high molecular polymer can mitigate the whole of cryoprotector by reducing the intracellular required protectant concentration of permeability
Body toxicity.In freezing, such protective agent is mainly used in combination with permeability protective agent, when cell being promoted to complete dehydration rewarming,
One hypertonic environment is provided, prevents moisture from entering cell and causes cell expansion to destroy fastly very much.Human serum albumins appropriate can
After playing similar cow's serum recovery, the effective component of cell in-vitro growth is maintained.Furthermore light hydroxyethyl starch not only low-temperature protection
Effect is good, and to human toxicity very little, adds it in preservation liquid and freeze marrow, not clean without eluting or eluting after rewarming,
It inputs in experimental animal body and is reacted without overt toxicity.
There is hypothesis to think that low temperature and freezing injury are related with the formation of oxygen radical, the formation of free radical will cause oxidation damage
Wound, such as peroxidatic reaction of lipid, protein oxidation and damage.Therefore, the factor of anti-apoptotic, Ke Yiti are added in cryopreservation
Motility rate after high cell recovery.The present invention is previously added Rock inhibitor Y27632 inhibitors of apoptosis when freezing, can be with
Intracorporal anti-apoptotic proteins of active cell, while can also promote cell metabolism and proliferation avoid inducing when cryopreservation recovery
Apoptosis.
The invention has the benefit that mesenchymal stem cell cryopreserving liquid of the invention is suitable for slow cryopreservation method, it can be with
The activity for maintaining mescenchymal stem cell for a long time, freezes and works well.
2. mesenchymal stem cell cryopreserving liquid of the invention reduces DMSO concentration and used serum substitute, answered from clinic
It is safer reliable with being seen with biological safety angle.
3. mesenchymal stem cell cryopreserving liquid definite ingredients of the invention, simple uncomplicated.
4. cell is living using mesenchymal stem cell cryopreserving liquid of the invention to recovering after mesenchymal stem cell cryopreserving 6 months
Rate is 90.7% or more.
Detailed description of the invention
Fig. 1 is the shadow grown according to the different frozen stock solutions of experimental example 1 provided by the invention to human umbilical cord mesenchymal stem cells
It rings；
Fig. 2 is the table of the umbilical cord mesenchymal stem cells frozen according to two kinds of frozen stock solutions of experimental example 1 provided by the invention
Type testing result comparison diagram, wherein Fig. 2-a is the cellular morphology comparison for the umbilical cord mesenchymal stem cells that two kinds of frozen stock solutions froze
Figure, Fig. 2-b are the flow cytometer detection phenotypic results comparison diagram for the umbilical cord mesenchymal stem cells that two kinds of frozen stock solutions froze；
Fig. 3 is dry to placenta mesenchyma according to the frozen stock solution containing different DMSO concentration of experimental example 2 provided by the invention
The influence of proliferation activity after cell recovery.
Fig. 4 is dry to placenta mesenchyma according to the frozen stock solution containing different DMSO concentration of experimental example 2 provided by the invention
The comparison diagram of Osteoblast Differentiation ability after cell recovery, wherein Fig. 4-a be DMSO concentration be 5% frozen stock solution to placenta mesenchyma
Osteoblast Differentiation ability after stem cell recovery；Fig. 4-b is that the frozen stock solution that DMSO concentration is 10% recovers to placenta mesenchyma stem cell
Osteoblast Differentiation ability afterwards.
Fig. 5 is dry to placenta mesenchyma according to the frozen stock solution containing different DMSO concentration of experimental example 2 provided by the invention
At the comparison diagram of rouge differentiation capability after cell recovery, wherein Fig. 5-a be DMSO concentration be 5% frozen stock solution to placenta mesenchyma
At rouge differentiation capability after stem cell recovery；Fig. 5-b is that the frozen stock solution that DMSO concentration is 10% recovers to placenta mesenchyma stem cell
Afterwards at rouge differentiation capability.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, technical solution of the present invention will be carried out below
Detailed description.Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Base
Embodiment in the present invention, those of ordinary skill in the art are obtained all without making creative work
Other embodiment belongs to the range that the present invention is protected.
Unless otherwise stated, the DMSO in the present invention is the English abbreviation of dimethyl sulfoxide, and HES is the English of hydroxyethyl starch
Literary referred to as HSE is the sero-abluminous English abbreviation of people, and FBS is the English abbreviation of fetal calf serum, and bFGF is that basic fibroblast is thin
The English abbreviation of the intracellular growth factor, AB indicate that people AB serum, MSCs cell are mescenchymal stem cell, and UC-MSCs is between people's umbilical cord
Mesenchymal stem cells, ROCK inhibitor Y27632 are Rock inhibitor Y27632, and PBS is 1 × phosphate buffer.
Embodiment 1
A kind of mesenchymal stem cell cryopreserving liquid is present embodiments provided, including dimethyl sulfoxide is 5%, human serum albumin is
10%, DMEM/F-12 culture medium are that 75%, Rock inhibitor Y27632 is 5umol/L and hydroxyethyl starch is 6%.Dimethyl
Sulfoxide is 5%, human serum albumin 10%, DMEM/F-12 culture medium be 75%, Rock inhibitor Y27632 be 5umol/L and
Hydroxyethyl starch is 6%.
The preparation method comprises the following steps: according to the frozen stock solution volume of cell density and quasi- configuration, first take corresponding proportion hydroxyethyl starch,
Rock inhibitor Y27632 and serum-free stem cell media, are gently suspended；Then the human seralbumin egg of corresponding ratio is added
It is white, gently it is suspended；Dimethyl sulfoxide is finally added dropwise, mixes, the as described mesenchymal stem cell cryopreserving liquid.
Application method are as follows: collect cell to be frozen, digest, supernatant is abandoned in centrifugation；Then to be frozen to treated thin
Sterile PBS is added in born of the same parents, cell is counted, is centrifuged again after counting；According to freezing density (5 × 106A/ml) it calculates and intends
The total volume of freeze-stored cell, according to the overall accumulated amount frozen, be first added hydroxyethyl starch (HES) and Rock inhibitor Y27632,
Serum-free stem cell media, is gently suspended；Then human serum albumins (HSE) is being added, is gently being suspended；Finally it is added dropwise
DMSO is finally mixed, and freezing is transferred in liquid nitrogen container and saved.
Embodiment 2
A kind of mesenchymal stem cell cryopreserving liquid is present embodiments provided, including dimethyl sulfoxide is 5%, human serum albumin is
10%, DMEM/F-12 culture medium are that 70%, Rock inhibitor Y27632 is 5umol/L and hydroxyethyl starch is 6%.
Preparation method is, according to the frozen stock solution volume of cell density and quasi- configuration, first take corresponding proportion hydroxyethyl starch,
Rock inhibitor Y27632 and serum-free stem cell media, are gently suspended；Then the human seralbumin egg of corresponding ratio is added
It is white, gently it is suspended；Dimethyl sulfoxide is finally added dropwise, mixes, dimethyl sulfoxide is put into 4 DEG C of refrigerators before dropwise addition and is pre-chilled
10-30min is pre-chilled to 4 DEG C, the as described mesenchymal stem cell cryopreserving liquid.
Application method are as follows: collect cell to be frozen, digest, supernatant is abandoned in centrifugation；Then to be frozen to treated thin
Sterile PBS is added in born of the same parents, cell is counted, is centrifuged again after counting；According to freezing density (2 × 106A/ml) it calculates and intends
The total volume of freeze-stored cell, according to the overall accumulated amount frozen, be first added hydroxyethyl starch (HES) and Rock inhibitor Y27632,
Serum-free stem cell media, is gently suspended；Then human serum albumins (HSE) is being added, is gently being suspended；Finally it is added dropwise
DMSO is finally mixed, and frozen stock solution containing cell is dispensed into cryopreservation tube, then cryopreservation tube is put into program temperature reduction box, so
Program temperature reduction box is put into -80 DEG C of refrigerators 6 hours or more afterwards, transfers in liquid nitrogen container and saves.It can be marked on cryopreservation tube
Cell Name, date and quantity etc..
Experimental example 1:
Study the influence of mesenchymal stem cell cryopreserving liquid of the invention to human umbilical cord mesenchymal stem cells cryopreservation resuscitation
1. experiment purpose: comparing and evaluate mesenchymal stem cell cryopreserving liquid of the invention and common frozen stock solution between people's umbilical cord
The effect of mesenchymal stem cells cryopreservation resuscitation
2. experimental method: this experimental method carries out after freezing 1,3,6 months UC-MSCs by using different frozen stock solutions
Recovery, be then respectively adopted trypan blue counting, FCM analysis phenotype, CCK8 analyze cell proliferative capacity, evaluate this two
Kind cells frozen storing liquid freezes effect to UC-MSCs.
The various experimental implementations being specifically related to are as follows:
(1) UC-MSCs being collected into: being inoculated with by cell culture processes by the density of 2000-5000/cm2 respectively,
Then be added suitable stem cell complete medium (are as follows: DMEM/F12+15%FBS+10 ng/mL bFGF+100U/mL mould
Plain+100 μ g/mL streptomysins) 37 DEG C are placed in, 5%CO2Incubator culture.(patch after the adherent adhesion area of born of the same parents is up to 70%~90%
When parietal cell grows to 70%-90% fusion), it is digested with 0.25% pancreatin, 200g is centrifuged 5min, supernatant is abandoned, then with dry nothing
Serum stem cell media is resuspended, and then carries out subsequent experimental.
(2) cell cryopreservation: the final cell density that this programme freezes is 1x106A/ml.
1. control group, C (uses conventional cryopreservation liquid): by the ratio of percent by volume DMSO:FBS:DMEM/F12=1:4:5
Example, frozen stock solution required for configuration, mixes in advance, is placed in 4 DEG C of refrigerators pre-cooling half an hour in advance, reduces DMSO in room temperature volatilization heat
Amount and toxicity lethal cell, being placed in 4 degree in advance can toxicity to avoid DMSO to cell.Then according to the cell being collected into
Quantity is added the frozen stock solution appropriate being pre-chilled in advance, mixes gently cell, make its density 2x106A/ml, respectively packing with
In cryopreservation tube, label is finally putting into program temperature reduction box, and -80 DEG C of placement 8h are stayed overnight, and is transferred in liquid nitrogen within second day and is saved.
2. experimental group, S (uses mesenchymal stem cell cryopreserving liquid of the invention): press percent by volume DMSO:HSE:
The ratio of DMEM/F12=1:2:7, according to cell density (2x106A/ml) and the quasi- frozen stock solution volume configured, first take corresponding ratio
The hydroxyethyl starch the 6% of total volume (additive amount be) of example, Rock inhibitor Y27632 (additive amount 5umol/L) and without blood
Clear stem cell media, is gently suspended；Then the human serum albumins for adding corresponding ratio, is gently suspended；Finally add dropwise
Enter dimethyl sulfoxide, mixes, the as described mesenchymal stem cell cryopreserving liquid, totality frozen stock solution ingredient when final are as follows: 5% (v/v)
The DMEM/F12 of DMSO+10% (v/v) HSE+6% (w/v) HES+5um Rock inhibitor Y27632.
(3) recovery of cell: cell is taken out from liquid nitrogen container, is quickly transferred to place 2min in 37 DEG C of water-baths, warp
It is inoculated into culture dish and cultivates after PBS washing.
(4) cell cell count and survival rate detection: is diluted to 2 × 10 with culture medium6It is thin to draw 10 μ l for/ml concentration
The 0.4% trypan blu e solution of born of the same parents' suspension and 10 μ l are added in loading glass slide after being sufficiently mixed, then thin using CountStar
Born of the same parents' calculating instrument count and survival rate detects.
(5) morphological observation: before cell jelly and after recovery for 24 hours, culture dish is taken out from incubator, is placed on inversion
Microscopically observation simultaneously photographs to record.
(6) cell growth curve: after cell recovery washing, cell is resuspended with appropriate culture medium, every hole adds into 96 orifice plates
Enter 0.2ml cell suspension (containing 200 living cells), places 37 DEG C, 5%CO2It is cultivated 7 days in incubator, fixed point is added daily
After 20ul CCK8 is cultivated 2 hours, the proliferative conditions of cell are detected, and draw growth curve.
(7) flow cytometer detection: after cell recovery culture to 80-90% degrees of fusion when carry out flow cytometer detection.What preparation had digested
For umbilical cord mesenchymal stem cells, antibody label is carried out to cell after PBS washing, labelled antibody is PE-CD73, PE- respectively
CD105, PE-CD90, PE-CD34, FITC-CD45, FITC-HLA-Dr, PE-CD44, PE-CD29, PE-Mouse IgG1 and
FITC-Mouse IgG1 (purchase from BD bioscience) by antibody.After being incubated for washing, upper machine testing.
3. experimental result:
The motility rate result such as table 1 of (1) two kind of different frozen stock solution recovery:
Influence of the different frozen stock solutions of table 1. to umbilical cord mesenchymal stem cells motility rate
Although 1 Cell viability of table the results show that two kinds of cell recovery motility rates of first trimester there was no significant difference, freeze
6th month as the result is shown: the frozen stock solution cell recovery motility rate of experimental group is higher, can be reduced Apoptosis.
(2) two kinds of cells frozen storing liquids are proliferated testing result such as Fig. 1: after UC-MSCs freezes 6 months in two kinds of frozen stock solutions,
It recovers, after being centrifuged and being resuspended, these cells is carried out observing cell after their cryopreservation resuscitations with isopycnic bed board respectively
Proliferative capacity, as a result, it has been found that: the UC-MSC ability of cell proliferation of mesenchymal stem cell cryopreserving liquid cryopreservation resuscitation of the invention is bright
The aobvious ability of cell proliferation for being better than control group.
(3) cell phenotype testing result such as Fig. 2: the UC-MSCs that both frozen stock solutions were frozen using flow cytometer
Phenotypic examination is carried out, after discovery freezes 6 months, the phenotypes of two kinds of cells there are no significant difference illustrates this frozen stock solution pair
The phenotype of MSCs may be applicable to freezing for mescenchymal stem cell without influence.
Experimental example 2:
The influence that the novel frozen stock solution of various concentration DMSO recovers to Human plactnta mesenchymal stem cell cryopreserving
1. experiment purpose: comparing and evaluate the mescenchymal stem cell of the invention of 5% concentration DMSO and 10% concentration DMSO
The effect that frozen stock solution recovers to Human plactnta mesenchymal stem cell cryopreserving.
2. experimental method: the frozen stock solution cryopreserved human placenta that this experimental method is configured by using 5% and 10% concentration DMSO
After mescenchymal stem cell 6 months, cell recovery is carried out, trypan blue counting is then respectively adopted, CCK8 analyzes the proliferation energy of cell
Power and differentiation capability evaluate this various concentration cells frozen storing liquid and freeze effect to placenta mesenchyma stem cell.It is specifically related to
The various experimental implementations arrived are specific as follows:
(1) cell culture processes: respectively by the placenta mesenchyma stem cell being collected by 2000-5000/cm2It is close
Degree be inoculated with, be then added suitable stem cell complete medium (are as follows: DMEM/ F12+15%FBS+10ng/mL bFGF+
+ 100 μ g/mL streptomysin of 100U/mL penicillin) 37 DEG C are placed in, 5%CO2Incubator culture.When the adherent adhesion area of born of the same parents reaches
It after 70%~90% (when adherent cell growth to 70-90% merges), is digested with 0.25% pancreatin, 200g centrifugation 5min, in abandoning
Clearly, it is then resuspended with dry serum-free stem cell media, then carries out subsequent experimental.
(2) cell cryopreservation: the final cell density that this programme freezes is 1x106A/ml, frozen stock solution configuration are dilute using half times
Interpretation of the law, so operating method is as follows:
1. containing the DMEM/F12 of 5% (v/v) DMSO+10% (v/v) HSE+6% (w/v) HES+5um Rock-Y27632
Frozen stock solution configuration: first according to the ratio of percent by volume DMSO:HSE:DMEM/F12=1:2:17；According to freeze density (2 ×
106A/ml) total volume that calculates quasi- freeze-stored cell, according to the overall accumulated amount frozen, hydroxyethyl starch is first added, and ((additive amount is
The 6% of total volume) and Rock inhibitor Y27632 (additive amount 5umol/L), serum-free stem cell media, gently it is suspended；
Then human serum albumins (HSE) is being added, is gently being suspended；DMSO is finally added dropwise, finally mixes.
2. containing the DMEM/F12 of 10% (v/v) DMSO+10% (v/v) HSE+6% (w/v) HES+5um Rock-Y27632
Frozen stock solution configuration: first according to the ratio of percent by volume DMSO:HSE:DMEM/F12=2:2:6, according to freezing density (2 × 106
A/ml) total volume for intending freeze-stored cell is calculated, according to the overall accumulated amount frozen, hydroxyethyl starch is first added, and ((additive amount is total
The 6% of volume) and Rock inhibitor Y27632 (additive amount 5umol/L), serum-free stem cell media, gently it is suspended；So
Human serum albumins (HSE) is being added afterwards, is gently being suspended；DMSO is finally added dropwise, finally mixes, as frozen stock solution.
(3) recovery of cell: cell is taken out from liquid nitrogen container, is quickly transferred to place 2min in 37 DEG C of water-baths, warp
It is inoculated into culture dish and cultivates after PBS washing.
(4) cell cell count and survival rate detection: is diluted to 2 × 10 with culture medium6A/ml concentration draws 10 μ l
The 0.4% trypan blu e solution of cell suspension and 10 μ l are added in loading glass slide after being sufficiently mixed, and then use CountStar
Cell counter count and survival rate detects.
(5) morphological observation: before cell jelly and after recovery for 24 hours, culture dish is taken out from incubator, is placed on inversion
Microscopically observation simultaneously photographs to record.
(6) cell growth curve: after cell recovery washing, cell is resuspended with appropriate culture medium, every hole adds into 96 orifice plates
Enter 0.2ml cell suspension (containing 200 living cells), places 37 DEG C, 5%CO2It is cultivated 7 days in incubator, fixed point is added daily
After 20ul CCK8 is cultivated 2 hours, the proliferative conditions of cell are detected, and draw growth curve.
(7) differentiation capability detects: after the cell culture after recovery to 80-90% degrees of fusion, had digestive transfer culture carries out skeletonization
Break up with lipoblast.Umbilical cord MSC is thin using the osteoblast differentiation and fat of Gibco to osteoblast and Adipocyte Differentiation
Born of the same parents' differentiation agents box, article No. are A10072-01, A10070-01 respectively.Experimental implementation by specification requires to carry out.
3. experimental result:
The motility rate result such as table 2 of (1) two kind of different frozen stock solution recovery:
Influence of the frozen stock solution of 2. various concentration DMSO of table to Human plactnta mescenchymal stem cell
As can be seen from Table 2, it recovers after cell cryopreservation 6 months, Cell viability testing result is shown, the group containing 5%DMSO
Cells frozen storing liquid motility rate is higher, and average motility rate is 90.7%；And average motility rate after the frozen stock solution cell recovery of 10%DMSO group are as follows:
86.47%.It can therefore be concluded that both formula frozen stock solutions are suitable for placenta mesenchyma stem cell, but reduction appropriate
DMSO concentration, can reduce DMSO can be to the toxic effect of cell, thus to improve the recovery motility rate of placenta mesenchyma stem cell.
(2) influence of the frozen stock solution containing different DMSO concentration to proliferation activity after placenta mesenchyma stem cell recovery is as schemed
3: the bed board with condition being carried out by the cell recovered to these, it is allowed to be proliferated.CCK8 is the results show that both differences DMSO
The frozen stock solution of concentration was in continuous culture seven days, statistical research discovery, proliferative capacity between the two without difference (P <
0.05)。
(3) the two kinds of influences of frozen stock solution to placenta stem-cell differentiation capability: where two kinds of frozen stock solutions to placenta stem-cell at
The comparison of bone differentiation capability such as Fig. 4, two kinds of frozen stock solutions are to placenta stem-cell at comparison such as Fig. 5 of rouge differentiation capability.Pass through difference
The placenta stem-cell that two kinds of frozen stock solutions are recovered after freezing carries out osteoblast differentiation and lipoblast Analytical Chemical Experiment, induction 21
After it, dyestuff is shown, both frozen stock solutions are on placenta stem-cell differentiation capability without influence.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any
Those familiar with the art in the technical scope disclosed by the present invention, can easily think of the change or the replacement, and should all contain
Lid is within protection scope of the present invention.Therefore, protection scope of the present invention should be based on the protection scope of the described claims.
Claims (10)
1. a kind of mesenchymal stem cell cryopreserving liquid, which is characterized in that the mesenchymal stem cell cryopreserving liquid includes Rock inhibitor
Y27632, dimethyl sulfoxide, human serum albumin, hydroxyethyl starch and serum-free stem cell media.
2. mesenchymal stem cell cryopreserving liquid according to claim 1, which is characterized in that the mesenchymal stem cell cryopreserving liquid
By percent by volume include dimethyl sulfoxide be 5-10%, human serum albumin 5-10%, serum-free stem cell media be 70-
90%, Rock inhibitor Y27632 are 5-10umol/L and hydroxyethyl starch is 3-6%.
3. mesenchymal stem cell cryopreserving liquid according to claim 2, which is characterized in that the mesenchymal stem cell cryopreserving liquid
By percent by volume include dimethyl sulfoxide be 5%, human serum albumin 10%, serum-free stem cell media be 75%,
Rock inhibitor Y27632 is 7.5umol/L and hydroxyethyl starch is 6%.
4. mesenchymal stem cell cryopreserving liquid according to any one of claims 1 to 3, which is characterized in that the serum-free is dry thin
Born of the same parents' culture medium is DMEM/F-12 culture medium.
5. the method for preparing any mesenchymal stem cell cryopreserving liquid of Claims 1-4, which is characterized in that the method
The following steps are included: first taking hydroxyethyl starch, Rock inhibitor Y27632 and serum-free stem cell media, gently it is suspended；So
After add human serum albumins, be gently suspended；Dimethyl sulfoxide is finally added dropwise, mixes, the as described mesenchyma is dry thin
Born of the same parents' frozen stock solution.
6. the preparation method of mesenchymal stem cell cryopreserving liquid according to claim 5, which is characterized in that the dimethyl is sub-
Sulfone is put into 4 DEG C of refrigerators before dropwise addition and 10-30min is pre-chilled.
7. the preparation method of mesenchymal stem cell cryopreserving liquid according to claim 5, which is characterized in that the dimethyl is sub-
The volume ratio 1:2:7 of sulfone, the human serum albumin and the serum-free stem cell media.
8. a kind of application method of any mesenchymal stem cell cryopreserving liquid of Claims 1-4, which is characterized in that described
Application method the following steps are included:
(1): first collecting cell to be frozen, digest, supernatant is abandoned in centrifugation；
(2): according to the cell density in frozen stock solution, be added into step (1) treated cell to be frozen hydroxyethyl starch,
Rock inhibitor Y27632 and serum-free stem cell media, are gently suspended；Then human serum albumins is added, is gently mixed
It is outstanding；Dimethyl sulfoxide is finally added dropwise, is freezed after mixing.
9. the application method of mesenchymal stem cell cryopreserving liquid according to claim 8, which is characterized in that in step (2), institute
Stating the cell density in frozen stock solution is 2-10 × 106A/ml.
10. the application method of mesenchymal stem cell cryopreserving liquid according to claim 8, which is characterized in that in step (2),
The freezing specifically: frozen stock solution containing cell is dispensed into cryopreservation tube, then cryopreservation tube is put into program temperature reduction box and is dropped
Then program temperature reduction box is put into -80 DEG C of refrigerators and freezes, transfers in liquid nitrogen container and save by temperature.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810982226.4A CN109090100A (en) | 2018-08-27 | 2018-08-27 | A kind of mesenchymal stem cell cryopreserving liquid and preparation method thereof and application method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810982226.4A CN109090100A (en) | 2018-08-27 | 2018-08-27 | A kind of mesenchymal stem cell cryopreserving liquid and preparation method thereof and application method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109090100A true CN109090100A (en) | 2018-12-28 |
Family
ID=64851337
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810982226.4A Pending CN109090100A (en) | 2018-08-27 | 2018-08-27 | A kind of mesenchymal stem cell cryopreserving liquid and preparation method thereof and application method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109090100A (en) |
Cited By (24)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109699634A (en) * | 2019-01-31 | 2019-05-03 | 和携科技(北京)有限公司 | A kind of Cryopreservation and method for resuscitation of mescenchymal stem cell |
| CN109845726A (en) * | 2019-02-27 | 2019-06-07 | 李冲杰 | A kind of mescenchymal stem cell Cryopreservation protective agent |
| CN110463688A (en) * | 2019-07-10 | 2019-11-19 | 安徽农业大学 | A kind of dilution and store method of sheep sperm |
| CN110468095A (en) * | 2019-09-05 | 2019-11-19 | 安徽中盛溯源生物科技有限公司 | A kind of renal epithelial cell and its preparation method and application |
| CN110612978A (en) * | 2019-09-26 | 2019-12-27 | 广东华夏健康生命科学有限公司 | Preservation solution for human menstrual blood and preparation method thereof |
| CN111418579A (en) * | 2020-04-13 | 2020-07-17 | 广东华夏健康生命科学有限公司 | Preservation method of adipose tissues, preservation solution of adipose tissues and preparation method of preservation solution |
| CN111789105A (en) * | 2019-04-09 | 2020-10-20 | 北京大学第三医院（北京大学第三临床医学院） | Application of amino acid cryopreservation liquid in stem cell cryopreservation |
| CN111990396A (en) * | 2020-09-09 | 2020-11-27 | 江苏育瑞康生物科技有限公司 | Refrigerating fluid for mesenchymal stem cells and preparation method thereof |
| CN112011504A (en) * | 2020-09-09 | 2020-12-01 | 江苏育瑞康生物科技有限公司 | Preparation method of placenta decidua mesenchymal stem cells |
| CN112753694A (en) * | 2020-12-25 | 2021-05-07 | 江苏艾尔康生物医药科技有限公司 | Cell cryopreservation liquid, freezing method for mesenchymal stem cells and application of cell cryopreservation liquid |
| CN113349196A (en) * | 2021-05-14 | 2021-09-07 | 宁夏医科大学 | Mammary gland stem cell cryopreservation liquid and preparation method and cryopreservation method thereof |
| CN113396894A (en) * | 2021-07-06 | 2021-09-17 | 南方医科大学南方医院 | Composite freezing medium suitable for unit hair follicle preservation and preparation method and application thereof |
| CN113854280A (en) * | 2021-09-06 | 2021-12-31 | 创芯国际生物科技(广州)有限公司 | Novel low-temperature preservation solution and preparation method and application thereof |
| CN114164171A (en) * | 2021-11-18 | 2022-03-11 | 上海爱萨尔生物科技有限公司 | Human umbilical cord mesenchymal stem cell culture medium, injection, preparation method and application of human umbilical cord mesenchymal stem cell culture medium and injection in preparation of medicine for treating cerebral apoplexy |
| CN114480268A (en) * | 2022-01-21 | 2022-05-13 | 深圳市茵冠生物科技有限公司 | Preparation method of human umbilical cord mesenchymal stem cells |
| CN114557337A (en) * | 2022-02-21 | 2022-05-31 | 大连博格林生物科技有限公司 | Protein-free non-programmed cryopreservation liquid for umbilical cord mesenchymal stem cells and preparation method thereof |
| CN114747566A (en) * | 2022-04-26 | 2022-07-15 | 深圳市茵冠生物科技有限公司 | Mesenchymal stem cell cryopreservation solution and cryopreservation method thereof |
| CN114762496A (en) * | 2021-01-11 | 2022-07-19 | 南京艾尔普再生医学科技有限公司 | Cardiac muscle cell freezing medium and its preparation method |
| CN114762495A (en) * | 2021-01-11 | 2022-07-19 | 南京艾尔普再生医学科技有限公司 | Myocardial cell cryopreservation method |
| CN114794088A (en) * | 2022-06-24 | 2022-07-29 | 北京细胞治疗集团有限公司 | Human pluripotent stem cell cryopreservation liquid and application thereof |
| CN114946836A (en) * | 2022-05-25 | 2022-08-30 | 成都诺医德医学检验实验室有限公司 | Micro-tissue serum-free freezing medium and application thereof |
| CN115227876A (en) * | 2022-07-28 | 2022-10-25 | 福建省海西细胞生物工程有限公司 | Preparation method of tissue engineering epidermis |
| CN115299434A (en) * | 2022-08-09 | 2022-11-08 | 广州明迅生物科技有限责任公司 | Cell cryopreservation liquid, cell cryopreservation and recovery method and application thereof |
| CN116491501A (en) * | 2023-06-28 | 2023-07-28 | 北京嘉士腾医学检验实验室有限公司 | Primary tissue freezing solution and freezing, resuscitating and digesting method thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101990888A (en) * | 2009-08-31 | 2011-03-30 | 深圳市北科生物科技有限公司 | Stem cell freezing medium and freezing method of stem cells |
| CN103083323A (en) * | 2013-02-21 | 2013-05-08 | 中国医学科学院阜外心血管病医院 | Application of fasudil hydrochloride in preparation of medicament for promoting in-vivo survival and repair of mesenchymal stem cells and preparation of fasudil hydrochloride |
| CN103583511A (en) * | 2012-09-03 | 2014-02-19 | 四川新生命干细胞科技股份有限公司 | Mesenchymal stem cell cryopreserved liquid and injection liquid |
| CN104830763A (en) * | 2015-05-29 | 2015-08-12 | 广州赛莱拉干细胞科技股份有限公司 | Application of Y-27632 in cultivation of mesenchymal stem cells and cultivation method of mesenchymal stem cells |
| CN105325402A (en) * | 2015-11-13 | 2016-02-17 | 黄林海 | Cryopreservation protective agent for vitrification cryopreservation of marrow mesenchymal stem cells |
| CN105394027A (en) * | 2016-01-18 | 2016-03-16 | 黄林海 | Method for cryopreserving mesenchymal stem cells with low cryo-damage |
| CN106386783A (en) * | 2016-06-24 | 2017-02-15 | 安徽未名细胞治疗有限公司 | A mesenchymal stem cell cryoprotectant |
| CN108048398A (en) * | 2017-12-23 | 2018-05-18 | 淮北智淮科技有限公司 | A kind of stem cell cryopreserving and method for resuscitation |
-
2018
- 2018-08-27 CN CN201810982226.4A patent/CN109090100A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101990888A (en) * | 2009-08-31 | 2011-03-30 | 深圳市北科生物科技有限公司 | Stem cell freezing medium and freezing method of stem cells |
| CN103583511A (en) * | 2012-09-03 | 2014-02-19 | 四川新生命干细胞科技股份有限公司 | Mesenchymal stem cell cryopreserved liquid and injection liquid |
| CN103083323A (en) * | 2013-02-21 | 2013-05-08 | 中国医学科学院阜外心血管病医院 | Application of fasudil hydrochloride in preparation of medicament for promoting in-vivo survival and repair of mesenchymal stem cells and preparation of fasudil hydrochloride |
| CN104830763A (en) * | 2015-05-29 | 2015-08-12 | 广州赛莱拉干细胞科技股份有限公司 | Application of Y-27632 in cultivation of mesenchymal stem cells and cultivation method of mesenchymal stem cells |
| CN105325402A (en) * | 2015-11-13 | 2016-02-17 | 黄林海 | Cryopreservation protective agent for vitrification cryopreservation of marrow mesenchymal stem cells |
| CN105394027A (en) * | 2016-01-18 | 2016-03-16 | 黄林海 | Method for cryopreserving mesenchymal stem cells with low cryo-damage |
| CN106386783A (en) * | 2016-06-24 | 2017-02-15 | 安徽未名细胞治疗有限公司 | A mesenchymal stem cell cryoprotectant |
| CN108048398A (en) * | 2017-12-23 | 2018-05-18 | 淮北智淮科技有限公司 | A kind of stem cell cryopreserving and method for resuscitation |
Non-Patent Citations (2)
| Title |
|---|
| 郭立达等: "《动物细胞分离培养》", 31 August 2015, 重庆大学出版社 * |
| 黄友章等: "骨髓细胞液氮保存21-25年后的细胞活力体外研究", 《中国实验血液学杂志》 * |
Cited By (31)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109699634A (en) * | 2019-01-31 | 2019-05-03 | 和携科技(北京)有限公司 | A kind of Cryopreservation and method for resuscitation of mescenchymal stem cell |
| CN109845726A (en) * | 2019-02-27 | 2019-06-07 | 李冲杰 | A kind of mescenchymal stem cell Cryopreservation protective agent |
| CN111789105A (en) * | 2019-04-09 | 2020-10-20 | 北京大学第三医院（北京大学第三临床医学院） | Application of amino acid cryopreservation liquid in stem cell cryopreservation |
| CN110463688A (en) * | 2019-07-10 | 2019-11-19 | 安徽农业大学 | A kind of dilution and store method of sheep sperm |
| CN110468095A (en) * | 2019-09-05 | 2019-11-19 | 安徽中盛溯源生物科技有限公司 | A kind of renal epithelial cell and its preparation method and application |
| CN110612978A (en) * | 2019-09-26 | 2019-12-27 | 广东华夏健康生命科学有限公司 | Preservation solution for human menstrual blood and preparation method thereof |
| CN111418579B (en) * | 2020-04-13 | 2021-05-18 | 广东华夏健康生命科学有限公司 | Preservation method of adipose tissues, preservation solution of adipose tissues and preparation method of preservation solution |
| CN111418579A (en) * | 2020-04-13 | 2020-07-17 | 广东华夏健康生命科学有限公司 | Preservation method of adipose tissues, preservation solution of adipose tissues and preparation method of preservation solution |
| CN112011504A (en) * | 2020-09-09 | 2020-12-01 | 江苏育瑞康生物科技有限公司 | Preparation method of placenta decidua mesenchymal stem cells |
| CN111990396A (en) * | 2020-09-09 | 2020-11-27 | 江苏育瑞康生物科技有限公司 | Refrigerating fluid for mesenchymal stem cells and preparation method thereof |
| CN112753694A (en) * | 2020-12-25 | 2021-05-07 | 江苏艾尔康生物医药科技有限公司 | Cell cryopreservation liquid, freezing method for mesenchymal stem cells and application of cell cryopreservation liquid |
| CN114762496A (en) * | 2021-01-11 | 2022-07-19 | 南京艾尔普再生医学科技有限公司 | Cardiac muscle cell freezing medium and its preparation method |
| CN114762495B (en) * | 2021-01-11 | 2023-11-07 | 南京艾尔普再生医学科技有限公司 | Myocardial cell cryopreservation method |
| CN114762496B (en) * | 2021-01-11 | 2023-11-07 | 南京艾尔普再生医学科技有限公司 | Myocardial cell frozen stock solution and preparation method thereof |
| CN114762495A (en) * | 2021-01-11 | 2022-07-19 | 南京艾尔普再生医学科技有限公司 | Myocardial cell cryopreservation method |
| CN113349196A (en) * | 2021-05-14 | 2021-09-07 | 宁夏医科大学 | Mammary gland stem cell cryopreservation liquid and preparation method and cryopreservation method thereof |
| CN113396894A (en) * | 2021-07-06 | 2021-09-17 | 南方医科大学南方医院 | Composite freezing medium suitable for unit hair follicle preservation and preparation method and application thereof |
| CN113854280A (en) * | 2021-09-06 | 2021-12-31 | 创芯国际生物科技(广州)有限公司 | Novel low-temperature preservation solution and preparation method and application thereof |
| CN114164171A (en) * | 2021-11-18 | 2022-03-11 | 上海爱萨尔生物科技有限公司 | Human umbilical cord mesenchymal stem cell culture medium, injection, preparation method and application of human umbilical cord mesenchymal stem cell culture medium and injection in preparation of medicine for treating cerebral apoplexy |
| CN114480268A (en) * | 2022-01-21 | 2022-05-13 | 深圳市茵冠生物科技有限公司 | Preparation method of human umbilical cord mesenchymal stem cells |
| CN114480268B (en) * | 2022-01-21 | 2024-01-30 | 深圳市茵冠生物科技有限公司 | Preparation method of human umbilical cord mesenchymal stem cells |
| CN114557337A (en) * | 2022-02-21 | 2022-05-31 | 大连博格林生物科技有限公司 | Protein-free non-programmed cryopreservation liquid for umbilical cord mesenchymal stem cells and preparation method thereof |
| CN114747566A (en) * | 2022-04-26 | 2022-07-15 | 深圳市茵冠生物科技有限公司 | Mesenchymal stem cell cryopreservation solution and cryopreservation method thereof |
| CN114946836A (en) * | 2022-05-25 | 2022-08-30 | 成都诺医德医学检验实验室有限公司 | Micro-tissue serum-free freezing medium and application thereof |
| CN114946836B (en) * | 2022-05-25 | 2024-06-04 | 成都诺医德医学检验实验室有限公司 | Micro-tissue serum-free frozen stock solution and application thereof |
| CN114794088A (en) * | 2022-06-24 | 2022-07-29 | 北京细胞治疗集团有限公司 | Human pluripotent stem cell cryopreservation liquid and application thereof |
| CN115227876A (en) * | 2022-07-28 | 2022-10-25 | 福建省海西细胞生物工程有限公司 | Preparation method of tissue engineering epidermis |
| CN115227876B (en) * | 2022-07-28 | 2023-08-29 | 福建省海西细胞生物工程有限公司 | Preparation method of tissue engineering epidermis |
| CN115299434A (en) * | 2022-08-09 | 2022-11-08 | 广州明迅生物科技有限责任公司 | Cell cryopreservation liquid, cell cryopreservation and recovery method and application thereof |
| CN115299434B (en) * | 2022-08-09 | 2023-11-21 | 广州明迅生物科技有限责任公司 | Cell cryopreservation liquid, cell cryopreservation and resuscitation method and application thereof |
| CN116491501A (en) * | 2023-06-28 | 2023-07-28 | 北京嘉士腾医学检验实验室有限公司 | Primary tissue freezing solution and freezing, resuscitating and digesting method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109090100A (en) | A kind of mesenchymal stem cell cryopreserving liquid and preparation method thereof and application method | |
| Jang et al. | Cryopreservation and its clinical applications | |
| Weng et al. | Dimethyl sulfoxide-free cryopreservation for cell therapy: A review | |
| US10472606B2 (en) | Cell preservation method for pluripotent stem cells | |
| US8481253B2 (en) | Cryopreservation of adipose tissue for the isolation of mesenchymal stem cells | |
| CN108112575B (en) | Cryoprotective agents, cryoprotective and cryopreserved compositions, uses thereof, and methods of cryopreservation | |
| JP4031044B2 (en) | Use of arabinogalactan in cell cryopreservation medium | |
| KR100328742B1 (en) | Method and package design for cryopreservation and storage of cultured tisse equivalents | |
| CN104145943B (en) | A kind of frozen protection liquid of people's umbilical cord China Tong Shi glue tissue and preparation and application thereof | |
| CN108617638B (en) | Tissue and/or cell cryopreservation protective solution and preparation and application thereof | |
| CN109845728B (en) | Clinical application-grade adipose tissue cryopreservation liquid and cryopreservation method | |
| KR101321144B1 (en) | Vitrification medium for stem cells and vitrification method for stem cells using the same | |
| Diaz-Dussan et al. | Trehalose-based polyethers for cryopreservation and three-dimensional cell scaffolds | |
| AU8060191A (en) | Cryopreservation of cultured epithelial sheets | |
| CN104560869B (en) | A kind of method for preparing chorion mescenchymal stem cell | |
| AU2009228141A1 (en) | Mehtod, system, and apparatus for hypothermic collection, storage, transport and banking of birth tissue | |
| KR101407355B1 (en) | A composition for cryopreservation of stem cells or primary cells comprising plant-derived human serum albumin, plant protein hydrolysate and lipid | |
| EP3556849A1 (en) | Mammalian cell cryopreservation liquid | |
| US5895745A (en) | Method of thawing cryopreserved cells | |
| Freitas-Ribeiro et al. | Long-term and short-term preservation strategies for tissue engineering and regenerative medicine products: state of the art and emerging trends | |
| EP0671877B1 (en) | Cryoprotective aqueous solutions useful for the preservation of in vitro cultured epithelial cells. | |
| CN108029679A (en) | A kind of frozen stock solution for being used to freeze mononuclearcell | |
| Halberstadt et al. | Corneal cryopreservation with dextran | |
| CN111374123B (en) | Use method of neutral amino acid as cell cryoprotectant and application thereof | |
| Mutsenko | Cryopreservation of mesenchymal stromal cells within tissue engineering approaches |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20181228 |
|
| RJ01 | Rejection of invention patent application after publication |