CN109845723A - A kind of Human plactnta amnion and chorial frozen solution and preparation method thereof - Google Patents
A kind of Human plactnta amnion and chorial frozen solution and preparation method thereof Download PDFInfo
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- CN109845723A CN109845723A CN201711241799.3A CN201711241799A CN109845723A CN 109845723 A CN109845723 A CN 109845723A CN 201711241799 A CN201711241799 A CN 201711241799A CN 109845723 A CN109845723 A CN 109845723A
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Abstract
The present invention provides a kind of Human plactnta amnion and chorial frozen solution and preparation method thereof; the main DMSO of the frozen solution, sodium dodecyl aminopropionitrile, carbomer microballoon and lecithin according to certain part by weight composition; frozen solution made of the mixture of sodium dodecyl aminopropionitrile, carbomer microballoon and lecithin is added in DMSO and is remarkably improved the time that the frozen solution freezes Human plactnta amnion and chorion, the vigor of long-term cell preservation for the frozen solution.
Description
Technical field
The invention belongs to stem cell cryopreserving technical field, in particular to a kind of Human plactnta amnion and chorial cryoprotection
Liquid and preparation method thereof.
Background technique
Amnion and chorion are two chief components of Human plactnta.Amnion has certain in the innermost layer of placenta
Elasticity, and have multipotency, after transplanting will not the anti-multipotential stem cell of inducing immune-rejection, can secretory nerve nutrition because
Son is alternatively arranged as the basilar memebrane of ophthalmologic operation；And the same stem cell rich in more differentiation potentials on chorion, it can
Applied in the injuries of tissues and organs reparation due to caused by aging and lesion and transplanting.
Although amnion and chorion have very wide application range, the preservation of its profound hypothermia is at the bottleneck applied.
It is formed in the prior art for Preserved amniotic and chorial preservation liquid by by fetal calf serum, dimethyl sulfoxide and Dextran 40
Frozen solution, which is kept for the time of cell viability shorter, is unable to satisfy the clinical needs saved for a long time, and
Not and fetal calf serum used in the protection liquid is expensive, and different manufacturers difference production batch and quality exist significantly
Difference, and then the quality of entire frozen solution can be made a significant impact, and then influence the cell recovery rate frozen.
Summary of the invention
In order to solve the above technical problem, the present invention provides a kind of Human plactnta amnion and chorial frozen solution and its
Preparation method, the frozen solution can be with long-term preservation Human plactnta amnion and chorions, and can long-term cell preservation vigor.
Specific technical solution of the present invention is as follows:
The present invention provides a kind of Human plactnta amnion and chorial frozen solution, and the frozen solution is mainly by following heavy
The group of amount number is grouped as:
Further to improve, the carbomer microballoon is prepared by following parts by weight ingredient:
Phosphatidylserine 2.5-4.3
Carbomer 0.5-0.8.
Dodecylamino third is added in Human plactnta amnion provided by the invention and chorial frozen solution in DMSO
Frozen solution made of the mixture of sour sodium, carbomer microballoon and lecithin is remarkably improved the frozen solution to Human plactnta
The time that amnion and chorion freeze, the vigor of long-term cell preservation.
Further to improve, the carbomer microballoon is prepared via a method which to obtain: by the card wave of 3/4 parts by weight
Nurse is dissolved in the water of 2 parts by weight, and carbomer aqueous solution is made；Liquid is added in the carbomer of 1/4 parts by weight and phosphatidylserine
It is uniformly mixed in body paraffin and forms oily phase, carbomer aqueous solution is added in oily phase, clipped machine emulsification, stratification is gone
Except atoleine, washing is dry to get carbomer microballoon.
It is further to improve, it further include the lauroyl ethylene glycol that parts by weight are 0.2-0.5 parts in the frozen solution
Amine and 1-2.5 parts of Sodium Glycinate.
The present invention can be into one by the way that the mixture of lauroyl ethylene glycol amine and Sodium Glycinate is added in frozen solution
Step improves frozen solution to Human plactnta amnion and chorial holding time, the motility rate after improving cell recovery, and may be used also
To improve the stability of frozen solution.
Further to improve, the frozen solution mainly consists of the following components in parts by weight:
Another aspect of the present invention provides the preparation method of a kind of Human plactnta amnion and chorial frozen solution, this method
Include the following steps:
S1: the preparation of carbomer microballoon:
The carbomer of 3/4 parts by weight is dissolved in the water of 2 parts by weight, carbomer aqueous solution is made；By the card of 1/4 parts by weight
Wave nurse and phosphatidylserine, which are added in atoleine to be uniformly mixed, forms oily phase, and carbomer aqueous solution is added in oily phase,
Clipped machine emulsification, stratification remove atoleine, and washing is dry to get carbomer microballoon；
S2: being placed in sodium dodecyl aminopropionitrile, carbomer microballoon and lecithin in mortar and grind, obtains ground and mixed
Milled mixtures and lecithin are dissolved in DMSO to get frozen solution by object.
Further to improve, the weight fraction ratio of card carbomer microballoon and atoleine is 3-5.1:18-32.
It is further to improve, the actual conditions of cutter emulsification are as follows: first time emulsifying rate 2500rpm, time 5min, it is quiet
Set 1min；Second of emulsifying rate 4500rpm, time 5min stand 5min；Third time emulsification 5000rpm, time 10min.
It is further to improve, the specific steps of grinding are as follows: sodium dodecyl aminopropionitrile is dissolved in the water of 2 times of weight,
It is placed in mortar and grinds 5min, grinding rate 500rpm, the carbomer microballoon of 1/3 parts by weight and the lecithin of 2/3 parts by weight is added
Rouge continues to grind 10min, grinding rate 300rpm, the carbomer microballoon of 2/3 parts by weight is added, and continues to grind 10min, grinding
Speed 200rpm adds the lecithin of 1/3 parts by weight, grinds 10min, grinding rate 500rpm, obtains milled mixtures.
The present invention can significantly improve work of the frozen solution to cell by the restriction to cryoprotection liquid and preparation method thereof
Power.
Dodecylamino third is added in Human plactnta amnion provided by the invention and chorial frozen solution in DMSO
Frozen solution made of the mixture of sour sodium, carbomer microballoon and lecithin is remarkably improved the frozen solution to Human plactnta
The time that amnion and chorion freeze, the vigor of long-term cell preservation.
Specific embodiment
Embodiment 1
Embodiment 2
Embodiment 3
Carbomer microballoon is prepared via a method which to obtain: the carbomer of 3/4 parts by weight is dissolved in the water of 2 parts by weight
In, carbomer aqueous solution is made；The carbomer of 1/4 parts by weight and phosphatidylserine are added in atoleine and are uniformly mixed shape
At oily phase, carbomer aqueous solution is added in oily phase, clipped machine emulsification, stratification removes atoleine, washs, does
It is dry to get carbomer microballoon.
Embodiment 4
The preparation method of the frozen solution:
S1: the preparation of carbomer microballoon:
The carbomer of 3/4 parts by weight is dissolved in the water of 2 parts by weight, carbomer aqueous solution is made；By the card of 1/4 parts by weight
Wave nurse and phosphatidylserine, which are added in atoleine to be uniformly mixed, forms oily phase, and carbomer aqueous solution is added in oily phase,
Clipped machine emulsification, first time emulsifying rate 2500rpm, time 5min stand 1min；Second of emulsifying rate 4500rpm, when
Between 5min, stand 5min；Third time emulsification 5000rpm, time 10min, stratification removes atoleine, washs, dry,
Up to carbomer microballoon；
S2: sodium dodecyl aminopropionitrile being dissolved in the water of 2 times of weight, is placed in mortar and is ground 5min, grinding rate
The carbomer microballoon of 1/3 parts by weight and the lecithin of 2/3 parts by weight is added in 500rpm, continues to grind 10min, grinding rate
The carbomer microballoon of 2/3 parts by weight is added in 300rpm, continues to grind 10min, grinding rate 200rpm adds 1/3 parts by weight
Lecithin, grind 10min, grinding rate 500rpm, obtain milled mixtures, milled mixtures and lecithin are dissolved in DMSO
In to get frozen solution.
Embodiment 5
Embodiment 6
Embodiment 7
The preparation method of the frozen solution:
S1: the preparation of carbomer microballoon:
The carbomer of 3/4 parts by weight is dissolved in the water of 2 parts by weight, carbomer aqueous solution is made；By the card of 1/4 parts by weight
Wave nurse and phosphatidylserine, which are added in atoleine to be uniformly mixed, forms oily phase, and carbomer aqueous solution is added in oily phase,
Clipped machine emulsification, first time emulsifying rate 2500rpm, time 5min stand 1min；Second of emulsifying rate 4500rpm, when
Between 5min, stand 5min；Third time emulsification 5000rpm, time 10min, stratification removes atoleine, washs, dry,
Up to carbomer microballoon；
S2: sodium dodecyl aminopropionitrile being dissolved in the water of 2 times of weight, is placed in mortar and is ground 5min, grinding rate
The carbomer microballoon of 1/3 parts by weight and the lecithin of 2/3 parts by weight is added in 500rpm, continues to grind 10min, grinding rate
The carbomer microballoon of 2/3 parts by weight is added in 300rpm, continues to grind 10min, grinding rate 200rpm adds 1/3 parts by weight
Lecithin, grind 10min, grinding rate 500rpm, obtain milled mixtures, by milled mixtures, lecithin, lauroyl second
Glycol amine and Sodium Glycinate are dissolved in DMSO to get frozen solution.
Reference examples 1
Reference examples 2
Reference examples 3
Reference examples 4
Reference examples 5
Reference examples 6
Reference examples 7
The preparation method of the frozen solution:
S1: the preparation of carbomer microballoon:
The carbomer of 3/4 parts by weight is dissolved in the water of 2 parts by weight, carbomer aqueous solution is made；By the card of 1/4 parts by weight
Wave nurse and phosphatidylserine, which are added in atoleine to be uniformly mixed, forms oily phase, and carbomer aqueous solution is added in oily phase,
Clipped machine emulsification, first time emulsifying rate 4000rpm, time 5min；Second of emulsifying rate 1500rpm, time 5min；The
2000rpm, time 5min are emulsified three times, and stratification removes atoleine, and washing is dry to get carbomer microballoon；
S2: sodium dodecyl aminopropionitrile being dissolved in the water of 2 times of weight, is placed in mortar and is ground 5min, grinding rate
The carbomer microballoon of 1/3 parts by weight and the lecithin of 2/3 parts by weight is added in 500rpm, continues to grind 10min, grinding rate
The carbomer microballoon of 2/3 parts by weight is added in 300rpm, continues to grind 10min, grinding rate 200rpm adds 1/3 parts by weight
Lecithin, grind 10min, grinding rate 500rpm, obtain milled mixtures, milled mixtures and lecithin are dissolved in DMSO
In to get frozen solution.
Reference examples 8
The preparation method of the frozen solution:
S1: the preparation of carbomer microballoon:
The carbomer of 3/4 parts by weight is dissolved in the water of 2 parts by weight, carbomer aqueous solution is made；By the card of 1/4 parts by weight
Wave nurse and phosphatidylserine, which are added in atoleine to be uniformly mixed, forms oily phase, and carbomer aqueous solution is added in oily phase,
Clipped machine emulsification, first time emulsifying rate 2500rpm, time 5min stand 1min；Second of emulsifying rate 4500rpm, when
Between 5min, stand 5min；Third time emulsification 5000rpm, time 10min, stratification removes atoleine, washs, dry,
Up to carbomer microballoon；
S2: sodium dodecyl aminopropionitrile being dissolved in the water of 2 times of weight, is placed in mortar and is ground 5min, grinding rate
The carbomer microballoon of 1/3 parts by weight and the lecithin of 1/3 parts by weight is added in 500rpm, continues to grind 10min, grinding rate
The carbomer microballoon of 2/3 parts by weight and the lecithin of 2/3 parts by weight is added in 200rpm, continues to grind 20min, grinding rate
500rpm obtains milled mixtures, milled mixtures and lecithin is dissolved in DMSO to get frozen solution.
Reference examples 9
Reference examples 10
Reference examples 11
Reference examples 12
The detection of vigor after the freezing and recover of test example amnion stem cell and chorion stem cell
Amnion stem cell and chorion stem cell are collected, the cryoprotection of embodiment 1-7 and reference examples 1-12 is respectively adopted
Liquid saves, and is placed in liquid nitrogen, respectively freeze 1 month, 2 months, 3 months, 6 months, 9 months and 12 months and take from liquid nitrogen
The amnion stem cell and chorion stem cell frozen out, is placed in 37 DEG C of water-bath, recovery 1-2min, with trypan blue staining into
Row cell count, calculates cell survival rate, and testing result is shown in Table 1.Cell survival rate=(amnion stem cell and chorion after recovery
Stem cell sum-freezes proamnion stem cell and chorion stem cell sum)/freeze proamnion stem cell and chorion stem cell
Sum.
The testing result of each embodiment of table 1 and reference examples frozen solution freeze-stored cell viability rate
As can be seen from the table, with CN102763642A (dimethyl sulfoxide, people AB blood plasma, Bomaili A injection volume ratio
It 1:3:6) compares, the frozen solution that 1-7 of the embodiment of the present invention is provided can be dry thin with long-term preservation amnion stem cell and chorion
Born of the same parents, after saving 12 months, cell survival rate is more than 87.3%, when showing that frozen solution provided by the invention can be used for long
Between Preserved amniotic stem cell and chorion stem cell, compared with Example 1, the frozen solution that embodiment 4-8 is provided can be into
One step was improved to holding time of amnion stem cell and chorion stem cell, after saving 12 months, cell survival rate still above
91.3% or more, the cell survival rate saved from reference examples, which can be seen that, to be replaced each ingredient in frozen solution,
The preparation method deleted or change frozen solution will affect the cell viability that frozen solution freezes and the time frozen.
Claims (9)
1. a kind of Human plactnta amnion and chorial frozen solution, which is characterized in that the frozen solution is mainly by following
The group of parts by weight is grouped as:
2. Human plactnta amnion as described in claim 1 and chorial frozen solution, which is characterized in that the carbomer is micro-
Ball is prepared by following parts by weight ingredient:
Phosphatidylserine 2.5-4.3
Carbomer 0.5-0.8.
3. Human plactnta amnion as claimed in claim 2 and chorial frozen solution, which is characterized in that the carbomer is micro-
Ball is prepared via a method which to obtain: the carbomer of 3/4 parts by weight being dissolved in the water of 2 parts by weight, carbomer water is made
Solution；The carbomer of 1/4 parts by weight and phosphatidylserine are added in atoleine to be uniformly mixed and form oily phase, by carbomer
Aqueous solution is added in oily phase, clipped machine emulsification, stratification, removes atoleine, and washing is dry micro- to get carbomer
Ball.
4. Human plactnta amnion as described in claim 1 and chorial frozen solution, which is characterized in that the cryoprotection
Further include in liquid parts by weight be 0.2-0.5 parts of lauroyl ethylene glycol amine and 1-2.5 parts of Sodium Glycinate.
5. Human plactnta amnion as claimed in claim 3 and chorial frozen solution, which is characterized in that the cryoprotection
Liquid mainly consists of the following components in parts by weight:
6. a kind of preparation method of Human plactnta amnion and chorial frozen solution described in claim 1, which is characterized in that
Described method includes following steps:
S1: the preparation of carbomer microballoon:
The carbomer of 3/4 parts by weight is dissolved in the water of 2 parts by weight, carbomer aqueous solution is made；By the carbomer of 1/4 parts by weight
It is added in atoleine to be uniformly mixed with phosphatidylserine and forms oily phase, carbomer aqueous solution is added in oily phase, through cutting
Machine emulsification is cut, stratification removes atoleine, and washing is dry to get carbomer microballoon；
S2: being placed in sodium dodecyl aminopropionitrile, carbomer microballoon and lecithin in mortar and grind, obtains milled mixtures,
Milled mixtures and lecithin are dissolved in DMSO to get frozen solution.
7. preparation method as claimed in claim 6, which is characterized in that the weight fraction ratio of carbomer microballoon and atoleine is
3-5.1:18-32.
8. preparation method as claimed in claim 6, which is characterized in that the actual conditions of cutter emulsification are as follows: emulsify for the first time
Speed 2500rpm, time 5min stand 1min；Second of emulsifying rate 4500rpm, time 5min stand 5min；For the third time
Emulsify 5000rpm, time 10min.
9. preparation method as claimed in claim 6, which is characterized in that the specific steps of grinding are as follows: by dodecylamino third
Sour sodium is dissolved in the water of 2 times of weight, is placed in mortar and is ground 5min, grinding rate 500rpm, and the carbomer of 1/3 parts by weight is added
The lecithin of microballoon and 2/3 parts by weight continues to grind 10min, grinding rate 300rpm, and the carbomer that 2/3 parts by weight are added is micro-
Ball continues to grind 10min, grinding rate 200rpm, adds the lecithin of 1/3 parts by weight, grinds 10min, grinding rate
500rpm obtains milled mixtures.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114667997A (en) * | 2022-04-14 | 2022-06-28 | 杭州百凌生物科技有限公司 | Buffer solution for immunohistochemical detection of quality control product and preparation method and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114667997A (en) * | 2022-04-14 | 2022-06-28 | 杭州百凌生物科技有限公司 | Buffer solution for immunohistochemical detection of quality control product and preparation method and application thereof |
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