CN109907036A - Cells frozen storing liquid, cell cryopreservation liquid and preparation method thereof and cell freezing method - Google Patents
Cells frozen storing liquid, cell cryopreservation liquid and preparation method thereof and cell freezing method Download PDFInfo
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Abstract
The present invention provides a kind of cells frozen storing liquid, cell cryopreservation liquid and preparation method thereof and cell freezing methods, using by weight, every 100 parts of cells frozen storing liquids include: the cells frozen storing liquid of the distilled water of 4~12 portions of trehaloses, 20~35 parts of human serum albumins, 50~75 part of 0.9% sodium chloride injection and surplus.The cells frozen storing liquid does not contain DMSO, animal source component or hydroxyethyl starch 130, and toxic side effect is low, and the ingredient of frozen stock solution is simple, at low cost；The high recovery motility rate of cell is maintained, does not influence the characteristic of cell and the proliferative capacity of cell, facilitates the quick application and popularization of cell cryopreservation technology.
Description
Technical field
The present invention relates to technical field of cell biology, in particular to a kind of cells frozen storing liquid, cells frozen storing liquid
Preparation method and cell freezing method.
Background technique
Stem cell and immunocyte technology are widely applied at present, and cell storage is the important ring in entire cell industrial chain
Section.Cell cryopreservation needs to prevent cell from freezing and being damaged, be dehydrated and absorb water by ice crystal in resuscitation process using protective agent
Damage.Most widely used protective agent is dimethyl sulfoxide (DMSO), it is a kind of permeability protective agent, can reduce cell ice
Point reduces the formation of ice crystal, mitigates free radical to cell damage, changes biomembrane and produce to electrolyte, drug, poisonous substance and metabolism
The permeability of object.But it studies have shown that there are certain toxic effects by DMSO, has an effect, causes with protein hydrophobic group
Protein denaturation has vascular toxicity and liver renal toxicity, and there are potential carcinogenic risks.DMSO can be very good freezing and
Cell is protected in resuscitation process, but the cell after recovering is cultivated, and could be incited somebody to action by the metabolizing cells of certain time
DMSO discharge, cell can just carry out next step application at this time.Because of this process, so that stem cell receives very in the application
Big limitation.
It generally to use fetal calf serum or calf serum as protective agent in cells frozen storing liquid, protect cell membrane, improve and freeze
Deposit the cell survival rate of resuscitation process.But crazy heifer disease virus may be contained in cow's serum, endotoxin, exotoxin, there are also potential
Unknown virus risk.
Trehalose is by two glucose molecules with a, a, 1, and 1- glycosidic bond constitutes nonreducing sugar, and self property is very
Stablize, protective film can be formed in cell surface, be effectively protected that protein molecule is indeformable, and the life for the body that sustains life
Process and biological characteristic.A kind of cells frozen storing liquid is disclosed in the earlier patent application CN201810979369X of our company,
Using trehalose instead DMSO and fetal calf serum, the toxicity of frozen stock solution is reduced.However, in above-mentioned cells frozen storing liquid, still
Contain hydroxyethyl starch 130.Currently, hydroxyethyl starch 130 is restricted in the use of some departments, it is since ethoxy forms sediment
There are certain side effects for 130/0.4 sodium chloride injection of powder:
1, blood: changeable clotting mechanism leads to transient prothrombin time, the partial thromboplastin time of activation
And cruor time extending.Shi Yike is widely applied to cause transient prolonged bleeding time.
2, liver: being repeatedly transfused in the patient of this medicine, there is the raised report of indirect bilirubin, and 96 small after final injection
Shi Huifu is normal.
3, allergic reaction: small number of patients may occur in which allergic reaction using this medicine, show as palpebral edema, nettle rash and heavy breathing
Asthma etc..Also show the symptom of similar moderate influenza, bradycardia or tachycardia, bronchial spasm, non cardiogenic pulmonary edema.
4, other: still see vomiting, salivary gland and parotid gland enlargement, edema of lower extremity etc..Large dosage is in use, due to dilution
Effect may cause blood constituent such as coagulation factor, plasma protein dilution and hematocrit decline.
As it can be seen that the cell frozen using the cells frozen storing liquid containing hydroxyethyl starch 130, further applying also can be by
To limitation.
Summary of the invention
The present invention provides a kind of cells frozen storing liquid, cell cryopreservation liquid and preparation method thereof and cell freezing methods, at least to solve
Certainly its jelly is influenced containing the big ingredient of the side effects such as DMSO or hydroxyethyl starch 130 in cells frozen storing liquid in the related technology
The problem of depositing the further application of cell.
In a first aspect, the present invention provides a kind of cells frozen storing liquids, and by weight, every 100 parts of cells frozen storing liquid packets
Include: 4~12 portions of trehaloses, 20~35 parts of human serum albumins, 50~75 part of 0.9% sodium chloride injection and surplus distilled water.
Optionally, by weight, every 100 parts of cells frozen storing liquids include: 6~10 portions of trehaloses, 25~30 parts of people's blood
The distilled water of albumin, 55~65 part of 0.9% sodium chloride injection and surplus.
Optionally, the distilled water described in the cells frozen storing liquid is 0 part.
Optionally, by weight, every 100 parts of cells frozen storing liquids include: 8 portions of trehaloses, 30 parts of human serum albumins,
62 part of 0.9% sodium chloride injection.
Optionally, by weight, every 100 parts of cells frozen storing liquids include: 8 portions of trehaloses, 27 parts of human serum albumins,
61 part of 0.9% sodium chloride injection and 4 parts of distilled water.
The beneficial effect that the cells frozen storing liquid of first aspect has toxic side effect low.
Second aspect, the present invention provides a kind of preparation methods of cells frozen storing liquid described in first aspect, comprising:
Trehalose is weighed, 0.9% sodium chloride injection, human serum albumin and distilled water are measured；
The trehalose is dissolved in 0.9% sodium chloride injection, and institute is added in the solution that dissolution obtains
Human serum albumin and the distilled water are stated, is stirred evenly, mixed liquor is obtained；
By mixed liquor biofilter degerming, the cells frozen storing liquid is obtained.
The beneficial effect that there is the preparation method of the cells frozen storing liquid of second aspect preparation to be simple and convenient to operate.
The third aspect, the present invention provides a kind of cell freezing methods, comprising:
Cell is taken out from culture bottle, and 0.9% sodium chloride solution is added, cell suspension is made；
Cell suspension 1100rpm is centrifuged 5min, abandons supernatant, cells frozen storing liquid is added, cryopreservation tube is moved into, makes cryopreservation tube
Middle cell concentration is 5~9 × 106 cell/mL；
Cryopreservation tube is put into rapidly programmed cooling instrument, starting ready-made program cools the temperature to -90 DEG C in 1h or so and freezes；
Cryopreservation tube is transferred in liquid nitrogen after freezing and is saved for a long time.
The cell freezing method of the third aspect has easy to operate, quick beneficial effect.
Cells frozen storing liquid, cell cryopreservation liquid and preparation method thereof and the cell freezing method provided through the embodiment of the present invention, is adopted
With by weight, every 100 parts of cells frozen storing liquids include: 4~12 portions of trehaloses, 20~35 parts of human serum albumins, 50~75
The cells frozen storing liquid of 0.9% sodium chloride injection of part and the distilled water of surplus, the cells frozen storing liquid do not contain DMSO, animal sources
Ingredient or hydroxyethyl starch 130, toxic side effect is low, and the ingredient of frozen stock solution is simple, at low cost；Maintain cell
High recovery motility rate, does not influence the characteristic of cell and the proliferative capacity of cell, facilitate cell cryopreservation technology it is quick application and
It promotes.
Specific embodiment
The feature and exemplary embodiment of various aspects of the invention is described more fully below, in order to make mesh of the invention
, technical solution and advantage be more clearly understood, with reference to embodiments, the present invention is explained in further detail.Ying Li
Solution, described herein the specific embodiments are only for explaining the present invention, is not intended to limit the present invention.For art technology
For personnel, the present invention can be implemented in the case where not needing some details in these details.Below to embodiment
Description just for the sake of being better understood to provide to the present invention by showing example of the invention.
It should be noted that, in this document, the terms "include", "comprise" or its any other variant be intended to it is non-
It is exclusive to include, so that the process, method, article or equipment for including a series of elements not only includes those elements,
It but also including other elements that are not explicitly listed, or further include for this process, method, article or equipment institute
Intrinsic element.In the absence of more restrictions, the element limited by sentence " including ... ", it is not excluded that including
There is also other identical elements in the process, method, article or equipment of the element.
Embodiment 1
A kind of cells frozen storing liquid includes by weight: 4~12 portions of trehaloses, 20~35 parts of human serum albumins, 50~75
The distilled water of part 0.9% sodium chloride injection and surplus.
Cells frozen storing liquid 1: trehalose 4%, human serum albumin 35%, 0.9% sodium chloride injection 50%, distilled water
11%；
Cells frozen storing liquid 2: trehalose 6%, human serum albumin 20%, 0.9% sodium chloride injection 65%, distilled water
9%；
Cells frozen storing liquid 3: trehalose 4%, human serum albumin 20%, 0.9% sodium chloride injection 75%, distilled water
1%；
Cells frozen storing liquid 4: trehalose 5%, human serum albumin 35%, 0.9% sodium chloride injection 60%, distilled water
0%；
Cells frozen storing liquid 5: trehalose 10%, human serum albumin 30%, 0.9% sodium chloride injection 55%, distilled water
5%；
Cells frozen storing liquid 6: trehalose 12%, human serum albumin 25%, 0.9% sodium chloride injection 60%, distilled water
3%；
Cells frozen storing liquid 7: trehalose 8%, human serum albumin 30%, 0.9% sodium chloride injection 62%, distilled water
0%；
Cells frozen storing liquid 8: trehalose 8%, human serum albumin 27%, 0.9% sodium chloride injection 61%, distilled water
4%.
Trehalose in the present embodiment is commercially available from sigma company, and specification is 100 grams/bottle.
Human serum albumin in the present embodiment is commercially available from Harbin Paisi Feike Biopharmaceutical Co., Ltd., rule
Lattice are 50mL:10g/ bottles.
Embodiment 2
The cells frozen storing liquid provided in embodiment 1 is prepared by following method:
Step 1, according to predetermined formula, trehalose is weighed, measures 0.9% sodium chloride injection, human serum albumin
And distilled water；
Step 2, the trehalose is dissolved in 0.9% sodium chloride injection, and in the solution that dissolution obtains
The human serum albumin and the distilled water is added, stirs evenly, obtains mixed liquor；
Step 3, by mixed liquor biofilter degerming, the cells frozen storing liquid is obtained.
Optionally, above-mentioned biofilter can be 0.22 μm of biofilter.
Embodiment 3
In the present embodiment, it is tested using human umbilical cord mesenchymal stem cells, the cell configured in embodiment 1 is frozen
Liquid storage is tested.Cell cryopreservation uses following steps:
Step 1, cell is taken out from culture bottle, and 0.9% sodium chloride solution is added, cell suspension is made；
Step 2, cell suspension 1100rpm is centrifuged 5min, abandons supernatant, cells frozen storing liquid is added, moved into cryopreservation tube, make
Cell concentration is 5~9 × 106 cell/mL in cryopreservation tube；
Step 3, cryopreservation tube is put into rapidly to programmed cooling instrument (Thermo Scientific Forma CryoMed
Controlled Rate Freezer 7451), starting ready-made program cools the temperature to -90 DEG C in 1h or so and freezes；
Step 4, cryopreservation tube is transferred in liquid nitrogen after freezing and is saved for a long time.
Cell recovery
Cell is removed from liquid nitrogen, is put into rapidly in 37-40 DEG C of water-bath, notices that cell liquid level has to soak completely
Upstream face is hereinafter, freeze nozzle higher than the water surface, quickly rocking melts cell in 1min, to there is grain of rice size in liquid
Ice crystal can take out from water-bath.Cell after recovery is added directly into corresponding culture medium and is cultivated.After recovery
Second day micro- sem observation cell, takes pictures.Cell is observed with 4X object lens, the adherent ratio > 90% of cell, in each visual field only
A small amount of non-attached cell, attached cell are fully extended, and form is uniform, cell no abnormality seen form.
Cell count after recovery
From the cell suspension after recovery, 20 microlitres are drawn, it is micro- that configured 0.08% trypan blue working solution 20 is added
It rises, mixes gently, prevent bubble from generating, inhale 20 microlitres, be added in dedicated numeration plate, use Countstar cell counter
Cell quantity and survival rate test are carried out, is repeated twice.Experimental result is as shown in table 1.
Table 1
Experimental result is frozen such as using the cells frozen storing liquid (abbreviation ethoxy frozen stock solution) containing hydroxyethyl starch 130
Shown in table 2.
Table 2
Using the cells frozen storing liquid (abbreviation DMSO frozen stock solution) containing DMSO and the cells frozen storing liquid of the embodiment of the present invention
Contrast and experiment is as shown in table 3, table 4.
Table 3
Table 4
It is above-mentioned the results showed that using the embodiment of the present invention cells frozen storing liquid, freeze before and recovery after, cell is total
Number, concentration, motility rate show that frozen stock solution of the invention effective protection can be done in frozen storage process there is no significant changes
Cell.Show that the present invention after removal DMSO, hydroxyethyl starch 130, animal source component is implemented by contrast and experiment
The cell cryopreservation effect of the cells frozen storing liquid of example is no less than other cells frozen storing liquids of the prior art, and the embodiment of the present invention
Cells frozen storing liquid toxic side effect is low, and the ingredient of frozen stock solution is simpler, and cost is lower；The high recovery motility rate of cell is maintained,
The characteristic of cell and the proliferative capacity of cell are not influenced, facilitate the quick application and popularization of cell cryopreservation technology.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any
Modification, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (7)
1. a kind of cells frozen storing liquid, which is characterized in that by weight, every 100 parts of cells frozen storing liquids include: 4~12 parts of seas
Algae sugar, 20~35 parts of human serum albumins, 50~75 part of 0.9% sodium chloride injection and surplus distilled water.
2. cells frozen storing liquid according to claim 1, which is characterized in that by weight, every 100 parts of cells frozen storing liquids
It include: the distillation of 6~10 portions of trehaloses, 25~30 parts of human serum albumins, 55~65 part of 0.9% sodium chloride injection and surplus
Water.
3. cells frozen storing liquid according to claim 1, which is characterized in that the distilled water described in the cells frozen storing liquid is
0 part.
4. cells frozen storing liquid according to claim 3, which is characterized in that by weight, every 100 parts of cells frozen storing liquids
It include: 8 portions of trehaloses, 30 parts of human serum albumins, 62 part of 0.9% sodium chloride injection.
5. cells frozen storing liquid according to claim 1, which is characterized in that by weight, every 100 parts of cells frozen storing liquids
It include: 8 portions of trehaloses, 27 parts of human serum albumins, 61 part of 0.9% sodium chloride injection and 4 parts of distilled water.
6. a kind of preparation method of cells frozen storing liquid described in any one of claims 1 to 5 characterized by comprising
Trehalose is weighed, 0.9% sodium chloride injection, human serum albumin and distilled water are measured；
The trehalose is dissolved in 0.9% sodium chloride injection, and the people is added in the solution that dissolution obtains
Blood albumin and the distilled water, stir evenly, obtain mixed liquor；
By mixed liquor biofilter degerming, the cells frozen storing liquid is obtained.
7. a kind of cell freezing method characterized by comprising
Cell is taken out from culture bottle, and 0.9% sodium chloride solution is added, cell suspension is made；
Cell suspension 1100rpm is centrifuged 5min, abandons supernatant, cells frozen storing liquid is added, cryopreservation tube is moved into, makes cell in cryopreservation tube
Concentration is 5~9 × 106A cell/mL；
Cryopreservation tube is put into rapidly programmed cooling instrument, starting ready-made program cools the temperature to -90 DEG C in 1h or so and freezes；
Cryopreservation tube is transferred in liquid nitrogen after freezing and is saved for a long time.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111979172A (en) * | 2020-08-31 | 2020-11-24 | 安徽惠恩生物科技股份有限公司 | Infusion solution for keeping cells active for longer time and preparation method thereof |
| CN112998009A (en) * | 2021-03-31 | 2021-06-22 | 北京益华生物科技有限公司 | NK cell cryopreservation liquid and preparation method and application thereof |
| CN114073249A (en) * | 2021-10-29 | 2022-02-22 | 上海原天生物科技有限公司 | Slow quick freezing storage method for human T lymphocytes |
| CN114073249B (en) * | 2021-10-29 | 2024-01-02 | 上海原天生物科技有限公司 | Slow quick freezing method for human T lymphocyte |
| CN117099771A (en) * | 2023-07-27 | 2023-11-24 | 江苏太平洋美诺克生物药业股份有限公司 | Cell cryopreservation solution for high-throughput cryopreservation of Chinese hamster ovary cells |
| CN117099771B (en) * | 2023-07-27 | 2024-02-13 | 江苏太平洋美诺克生物药业股份有限公司 | Cell cryopreservation solution for high-throughput cryopreservation of Chinese hamster ovary cells |
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