CN107183008A - A kind of placenta mesenchyma stem cell frozen stock solution and its cryopreservation methods - Google Patents
A kind of placenta mesenchyma stem cell frozen stock solution and its cryopreservation methods Download PDFInfo
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- CN107183008A CN107183008A CN201710391992.9A CN201710391992A CN107183008A CN 107183008 A CN107183008 A CN 107183008A CN 201710391992 A CN201710391992 A CN 201710391992A CN 107183008 A CN107183008 A CN 107183008A
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- 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
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- 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
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
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Abstract
The present invention relates to stem cells technology field, more particularly to a kind of placenta mesenchyma stem cell frozen stock solution, in parts by weight including following components：Include following component in parts by weight：0.5 1.5 parts of 40 70 parts of DEME culture mediums, 26 parts of phosphoglycerol sodium solution, 15 parts of HES, 14 parts of mannitol solution, 0.3 0.8 parts of ethylene glycol, 0.5 2 parts of polyvinyl alcohol, 0.5 1.8 parts of trehalose and polyvinylpyrrolidone.Embryonic mesenchymal stem cells are preserved using the preservation liquid in the present invention, can alleviate during freezing to the damage caused by stem cell, improve the survival rate after stem cell recovery.
Description
Technical field
The present invention relates to stem cells technology field, more particularly to a kind of placenta mesenchyma stem cell frozen stock solution and its side of freezing
Method.
Background technology
Stem cell is the multipotential cell that a class has the of self-replication capacity.Under certain condition, it can be divided into many
Plant functioning cell.Stage of development according to residing for stem cell is divided into embryonic stem cell (embryonic stem cell, ES cell)
With adult stem cell (somatic stem cell).It is divided into three classes according to the potentiality of development of stem cell:Myeloid-lymphoid stem cell
(totipotent stem cell, TSC), multipotential stem cell (pluripotent stem cell) and unipotent stem cell
(unipotent stem cell) (specially energy stem cell).Stem cell (Stem Cell) is a kind of inabundant differentiation, still immature
Cell, with the potential function for regenerating various histoorgans and human body, medical field is referred to as " general-purpose cell ".
Contain a variety of stem cells, including mescenchymal stem cell, candidate stem cell, endothelial stem cell and unknown function in placenta
Pluripotent stem cell etc..Mescenchymal stem cell, English name is mesenchymal stem cells (MSC), also there is other
Title, such as marrow stromal cell, fat stem cell, pluripotency stroma cell, refer to that single or a group meets international uniform mark
Accurate cell.Placenta mesenchyma stem cell is an important kind in stem cell, and inside and outside is respectively provided with to bone, cartilage and fat
The ability of differentiation is organized, with the effect such as immunological regulation, hematopoiesis support, angiogenesis promoting.
Stem-cell therapy is after the stem cell with continuous self-reproduction ability and multidirectionalization potential is cultivated in vitro, then
Be implanted into that patient is impaired or defective tissue in, so as to realize the supplement to injury tissue and reparation.Current stem cell is main
Preserved using the mode of freezing, but the damage of stem cell can be caused during freezing, it is therefore desirable to add frozen stock solution to subtract
Slow cell damage.Existing frozen stock solution component includes dimethyl sulfoxide (DMSO), albumin and 1640 cultures rich in multiple nutritional components
Base, dimethyl sulfoxide (DMSO) is as a kind of permeability protective agent, with stronger penetration capacity, it is possible to decrease cell freezing point, reduction freezing
During damage suffered by cell, but dimethyl sulfoxide (DMSO) is poisonous, and proportion is big in frozen stock solution, easily causes albumen
Qualitative change, the survival rate of final influence stem cell.Frozen it is therefore desirable to develop a kind of frozen stock solution, and using the frozen stock solution
Deposit the cryopreservation methods of stem cell.
The content of the invention
The technical problems to be solved by the invention are：It is dry thin there is provided a kind of embryonic mesenchymal in view of the shortcomings of the prior art
Born of the same parents' frozen stock solution, the frozen stock solution can slow down freeze during damage suffered by mescenchymal stem cell, while also solving existing freeze
Liquid improves to the toxic effect of mescenchymal stem cell and freezes effect, improves the survival rate of mescenchymal stem cell.
In order to solve the above technical problems, the technical scheme is that：
A kind of embryonic mesenchymal stem cells frozen stock solution, in parts by weight including following component：DEME culture mediums 40-70
Part, 2-6 parts of phosphoglycerol sodium solution, 1-5 parts of HES, 1-4 parts of mannitol solution, 0.3-0.8 parts of ethylene glycol, polyethylene
0.5-1.5 parts of 0.5-2 parts of alcohol, 0.5-1.8 parts of trehalose and polyvinylpyrrolidone.
As an improvement technical scheme, the embryonic mesenchymal stem cells frozen stock solution includes in parts by weight
Following component：50-70 parts of DEME culture mediums, 4-6 parts of phosphoglycerol sodium solution, 3-5 parts of HES, mannitol solution 2-4
Part, 0.5-0.8 parts of ethylene glycol, 1-2 parts of polyvinyl alcohol, 0.8-1.5 parts of trehalose and 0.8-1.5 parts of polyvinylpyrrolidone.
As an improvement technical scheme, the embryonic mesenchymal stem cells frozen stock solution also include 1-3 parts of collagen.
As an improvement technical scheme, the mass concentration of the phosphoglycerol sodium solution is 0.5g/L-1.8g/L.
As an improvement technical scheme, the mass concentration of the mannitol solution is 0.2-1.5g/ml.
Another technical problem to be solved by this invention is：There is provided filled between a kind of embryo in view of the shortcomings of the prior art
The cryopreservation methods of matter stem cell.
In order to solve the above technical problems, the technical scheme is that：
A kind of cryopreservation methods of embryonic mesenchymal stem cells, the cryopreservation methods comprise the following steps：
(1) preparation of frozen stock solution：By the mannitol solution weighed, phosphoglycerol sodium solution, ethylene glycol and polyvinyl alcohol, fill
Divide well mixed, sequentially add DEME culture mediums, trehalose, collagen and polyvinylpyrrolidone, be sufficiently mixed, frozen
Liquid；
(2) placenta tissue is pre-processed：By the fresh human placenta tissue of acquisition, rinse removing bloodstain repeatedly with PBS, use alcohol swab
Ball sterilization placenta surface, then with normal saline flushing 2-3 times, it is standby；
(3) separation of placenta mesenchyma stem cell：The placenta tissue in step (2) is taken, fragment is cut into and is placed in centrifuge tube
In, isometric digestive ferment is added, is digested under the conditions of 30-37 DEG C, digestion collects digestive juice after terminating, is centrifuged, is received
Collect cell precipitation, obtain placenta mesenchyma stem cell；
(4) culture of placenta mesenchyma stem cell：Placenta mesenchyma stem cell in step (3) is inoculated with and trained
Support, the placenta mesenchyma stem cell in growth period of taking the logarithm, in the frozen stock solution that step (1) is inserted after cleaning, fully mix, obtain tire
The mixture of disk mescenchymal stem cell and frozen stock solution；
(5) placenta mesenchyma stem cell freezes：The mixture in step (4) is taken to place 10-30min on ice bath, then
Mixture is cooled stage by stage, the mixture after cooling stage by stage is finally inserted into freezen protective in liquid nitrogen.
As an improvement technical scheme, the digestive ferment includes trypsase, clostridiopetidase A and hyaluronidase, and institute
Trypsase, clostridiopetidase A and hyaluronidase are stated according to 0.1~0.7:0.3~0.8:0.2~0.5 volume ratio composition.
As an improvement technical scheme, cooling method is specially the first stage in -10 DEG C~-20 DEG C progress stage by stage
Cooling, second stage is cooled at -25 DEG C~-40 DEG C, and the phase III is cooled at -80 DEG C.
Employ after above-mentioned technical proposal, the beneficial effects of the invention are as follows：
(1) DEME culture mediums are used, for freezing and the recover amino acid needed for providing and battalion for embryonic mesenchymal stem cells
Form point；Mannitol solution can effectively deviate from intracellular moisture, reduce the formation of ice crystal；Phosphoglycerol sodium solution, hydroxyl second
Base starch, the mutual cooperation of ethylene glycol, polyvinyl alcohol, trehalose, polyvinylpyrrolidone and collagen, form stable liquid
System, the System forming membranaceous material coats embryonic mesenchymal stem cells by membranaceous material, frozen stock solution is between profit embryo is invaded
While mesenchymal stem cells, protective effect is also acted as, the damage frozen suffered by process embryonic mesenchymal stem cells is reduced, significantly
Improve the survival rate of embryonic mesenchymal stem cells；
(2) using above-mentioned frozen stock solution come freeze-stored cell, and in cell cryopreservation, with ice bath to the cold first, then use and divide
The stage mode of cooling carrys out freeze-stored cell, is effectively reduced the formation of cell interior ice crystal, and further reduction cell damage is hindered
Possibility.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Embodiment 1
A kind of embryonic mesenchymal stem cells frozen stock solution, in parts by weight including following component：45 parts of DEME culture mediums,
3 parts of phosphoglycerol sodium solution (mass concentration 0.5g/L), 2 parts of HES, 2 parts of (mass concentration 0.5g/ of mannitol solution
L), 0.5 part of 0.3 part of ethylene glycol, 0.5 part of polyvinyl alcohol, 0.5 part of trehalose and polyvinylpyrrolidone.
Embodiment 2
A kind of embryonic mesenchymal stem cells frozen stock solution, in parts by weight including following component：55 parts of DEME culture mediums,
4 parts of phosphoglycerol sodium solution (mass concentration 1g/L), 3 parts of HES, 3 parts of mannitol solution (mass concentration 0.8g/L),
0.8 part of 0.5 part of ethylene glycol, 1 part of polyvinyl alcohol, 0.8 part of trehalose, 2 parts of collagen and polyvinylpyrrolidone.
Embodiment 3
A kind of embryonic mesenchymal stem cells frozen stock solution, 68 parts of DEME culture mediums, 6 parts of (mass concentrations of phosphoglycerol sodium solution
1.5g/L), 5 parts of HES, 4 parts of mannitol solution (mass concentration 1.2g/L), 0.8 part of ethylene glycol, 2 parts of polyvinyl alcohol,
1.5 parts of 1.5 parts of trehalose, 2.5 parts of collagen and polyvinylpyrrolidone.
Embodiment 4
A kind of cryopreservation methods of embryonic mesenchymal stem cells, the cryopreservation methods comprise the following steps：
(1) preparation of frozen stock solution：By the mannitol solution weighed, ethylene glycol and polyvinyl alcohol, it is sufficiently mixed uniformly, then according to
Secondary addition DEME, trehalose, collagen and polyvinylpyrrolidone, are sufficiently mixed, obtain frozen stock solution；
(2) placenta tissue is pre-processed：By the fresh human placenta tissue of acquisition, rinse removing bloodstain repeatedly with PBS, use alcohol swab
Ball sterilization placenta surface, then with normal saline flushing 2-3 times, it is standby；
(3) separation of placenta mesenchyma stem cell：The placenta tissue in step (2) is taken, fragment is cut into and is placed in centrifuge tube
In, adding isometric digestive ferment, (trypsase, clostridiopetidase A and hyaluronidase are according to 0.4:0.3:0.2 volume ratio group
Into), digested, digestion collects digestive juice after terminating, centrifuged under the conditions of 30-37 DEG C, collect cell precipitation, obtained between placenta
Mesenchymal stem cells；
(4) culture of placenta mesenchyma stem cell：Placenta mesenchyma stem cell in step (3) is inoculated with and trained
Support, the placenta mesenchyma stem cell in growth period of taking the logarithm, in the frozen stock solution that step (1) is inserted after cleaning, fully mix, obtain tire
The mixture of disk mescenchymal stem cell and frozen stock solution；
(5) placenta mesenchyma stem cell freezes：The mixture in step (4) is taken to place 10-30min on ice bath, then
Mixture is frozen using cooling method stage by stage, the wherein first stage is cooled at -10 DEG C~-20 DEG C, second-order
Section is cooled at -25 DEG C~-40 DEG C, and the phase III is cooled at -80 DEG C.
Comparative example 1
A kind of embryonic mesenchymal stem cells frozen stock solution, compared with Example 3, unique difference are not contain in frozen stock solution sweet
Oleophosphoric acid sodium solution.
Comparative example 2
A kind of embryonic mesenchymal stem cells frozen stock solution, compared with Example 3, difference are in frozen stock solution not containing mannitol
Solution.
Comparative example 3
A kind of embryonic mesenchymal stem cells frozen stock solution, compared with Example 3, difference are in frozen stock solution not containing collagen.
Comparative example 4
A kind of embryonic mesenchymal stem cells frozen stock solution, compared with Example 3, difference are in frozen stock solution not containing polyethylene
Pyrrolidones.
Comparative example 5
A kind of embryonic mesenchymal stem cells frozen stock solution, compared with Example 3, difference are in frozen stock solution not containing polyethylene
Alcohol.
The embryonic mesenchymal stem cells that same batch is obtained are equally divided into 6 groups, and implementation is respectively adopted in every group of 20 samples
Component in example 1-3 and comparative example 1-3 configures frozen stock solution, and carries out freezing experiment, and it is 6 months to freeze the time, freezes knot
Shu Hou, recovers in 37 DEG C of water-baths, observe the precipitation of sample, and expects that orchid refuses dye method and determines every group of embodiment sample using platform
Survival rate, record data is simultaneously averaging processing, and result of the test is shown in Table 1.
Table 1 is embodiment 1-3 and the platform of comparative example 1-3 samples expects that orchid refuses dye rate and precipitation status
| Test example | Platform expects that orchid refuses dye rate | Precipitation status |
| Embodiment 1 | 96.2±0.5 | Nothing |
| Embodiment 2 | 97.3±1.2 | Nothing |
| Embodiment 3 | 97.6±1.5 | Nothing |
| Comparative example 1 | 42.3±1.2 | Precipitation is less |
| Comparative example 2 | 40.6±1.5 | Precipitation is less |
| Comparative example 3 | 56.8±1.5 | Precipitation is less |
| Comparative example 4 | 43.6±1.3 | Precipitation is less |
As shown in Table 1, the frozen stock solution of embodiment 1-3 configurations freezes to mescenchymal stem cell, the mesenchyma after recovery
The platform of stem cell expects that orchid refuses dye rate height, illustrates that mescenchymal stem cell died after recovery is less, illustrates phosphoglycerol sodium solution, hydroxyl second
Base starch, mannitol solution, ethylene glycol, polyvinyl alcohol, the mutual cooperation of trehalose and polyvinylpyrrolidone, form stable
Liquid system, the System forming membranaceous material serves certain effect to the survival rate of mescenchymal stem cell.And comparative example 1-4 matches somebody with somebody
The frozen stock solution put freezes to mescenchymal stem cell, and it is low that the platform of the mescenchymal stem cell after recovery expects that orchid refuses dye rate, between explanation
Mesenchymal stem cells died after recovery is more, illustrate without phosphoglycerol sodium solution, mannitol solution, polyvinyl alcohol, HES,
Collagen, the frozen stock solution of polyvinylpyrrolidone component freeze mescenchymal stem cell, can reduce the survival rate of mescenchymal stem cell.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
Any modifications, equivalent substitutions and improvements made within refreshing and principle etc., should be included in the scope of the protection.
Claims (8)
1. a kind of placenta mesenchyma stem cell frozen stock solution, it is characterised in that：Include following component in parts by weight：DEME is trained
Support 40-70 parts of base, 2-6 parts of phosphoglycerol sodium solution, 1-5 parts of HES, 1-4 parts of mannitol solution, ethylene glycol 0.3-0.8
Part, 0.5-2 parts of polyvinyl alcohol, 0.5-1.8 parts of trehalose and 0.5-1.5 parts of polyvinylpyrrolidone.
2. a kind of placenta mesenchyma stem cell frozen stock solution according to claim 1, it is characterised in that:In parts by weight
Including following component：50-70 parts of DEME culture mediums, 4-6 parts of sodium glycero-phosphate, 3-5 parts of HES, 2-4 parts of mannitol, second
0.8-1.5 parts of 0.5-0.8 parts of glycol, 1-2 parts of polyvinyl alcohol, 0.8-1.5 parts of trehalose and polyvinylpyrrolidone.
3. a kind of placenta mesenchyma stem cell frozen stock solution according to claim 1, it is characterised in that:The placenta mesenchyma
Stem cell cryopreserving liquid also includes 1-3 parts of collagen.
4. a kind of placenta mesenchyma stem cell frozen stock solution according to claim 1, it is characterised in that:The sodium glycero-phosphate
The mass concentration of solution is 0.5g/L-1.8g/L.
5. a kind of placenta mesenchyma stem cell frozen stock solution according to claim 1, it is characterised in that:The mannitol solution
Mass concentration be 0.2-1.5g/ml.
6. a kind of frozen stock solution using described in claim 1 freezes the cryopreservation methods of placenta mesenchyma stem cell, its feature exists
Comprise the following steps in the cryopreservation methods：
(1) preparation of frozen stock solution：It is fully mixed by the mannitol solution weighed, phosphoglycerol sodium solution, ethylene glycol and polyvinyl alcohol
Close uniform, sequentially add DEME culture mediums, trehalose, collagen and polyvinylpyrrolidone, be sufficiently mixed, obtain frozen stock solution；
(2) placenta tissue is pre-processed：By the fresh human placenta tissue of acquisition, rinse removing bloodstain repeatedly with PBS, disappeared with cotton ball soaked in alcohol
Malicious placenta surface, then with normal saline flushing 2-3 times, it is standby；
(3) separation of placenta mesenchyma stem cell：The placenta tissue in step (2) is taken, fragment is cut into and is placed in centrifuge tube, then
Isometric digestive ferment is added, is digested under the conditions of 30-37 DEG C, digestion collects digestive juice after terminating, is centrifuged, cell is collected
Precipitation, obtains placenta mesenchyma stem cell；
(4) culture of placenta mesenchyma stem cell：Placenta mesenchyma stem cell in step (3) is inoculated with and cultivated, is taken
In the frozen stock solution that step (1) is inserted after the placenta mesenchyma stem cell of exponential phase, cleaning, fully mix, obtain between placenta
The mixture of mesenchymal stem cells and frozen stock solution；
(5) placenta mesenchyma stem cell freezes：The mixture in step (4) is taken to place 10-30min on ice bath, then will be mixed
Compound is frozen using cooling method stage by stage, and the mixture after cooling stage by stage finally is inserted into freezen protective in liquid nitrogen.
7. a kind of cryopreservation methods that placenta mesenchyma stem cell is frozen using frozen stock solution according to claim 6, it is special
Levy and be:The digestive ferment includes trypsase, clostridiopetidase A and hyaluronidase, and the trypsase, clostridiopetidase A and transparent
Matter acid enzyme is according to 0.1~0.7:0.3~0.8:0.2~0.5 volume ratio composition.
8. a kind of cryopreservation methods that placenta mesenchyma stem cell is frozen using frozen stock solution according to claim 6, it is special
Levy and be:Cooling method is specially and cooled at -10 DEG C~-20 DEG C the first stage stage by stage, second stage -25 DEG C~-
40 DEG C are cooled, and the phase III is cooled at -80 DEG C.
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