CN109601529A - A kind of cell cryopreservation and method for resuscitation of simplicity - Google Patents
A kind of cell cryopreservation and method for resuscitation of simplicity Download PDFInfo
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- CN109601529A CN109601529A CN201910136226.7A CN201910136226A CN109601529A CN 109601529 A CN109601529 A CN 109601529A CN 201910136226 A CN201910136226 A CN 201910136226A CN 109601529 A CN109601529 A CN 109601529A
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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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- 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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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
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Abstract
The invention discloses the cell cryopreservations and method for resuscitation of a kind of simplicity, wherein cell cryopreservation is the following steps are included: preparing cell suspension and being centrifuged, cell precipitation is resuspended with cells frozen storing liquid, then it is transferred completely into cell cryopreservation tube, sealed membrane sealing is transferred in ultra low temperature freezer after successively pre-cooled and freezing and saves；The method of cell recovery is shaken, cell suspension is transferred in the culture bottle equipped with fresh culture immediately after melting completely and cultivates by frozen stock solution in 37-40 DEG C of water-bath the following steps are included: take out cell cryopreservation tube from -80 DEG C of ultra low temperature freezers.This method freeze-stored cell reduces the requirement to conditions of cryopreservation, under conditions of no liquid nitrogen, normal cell cryopreservation and recovery can be still carried out, duration is saved under conditions of not influencing cell activity up to 9 months, is able to satisfy requirement of the common laboratory to cell cryopreservation and recovery.
Description
Technical field
The invention belongs to cell technology application fields, and in particular to a kind of cell cryopreservation and method for resuscitation of simplicity.
Background technique
Cell is the basic unit for constituting body, is the basic unit of vital movement.Cell have it is independent, orderly from
Control metabolism system.It therefore, is the key that open life secret, conquer a stubborn disease, keep fit to the further investigation of cell.Currently,
Cell culture technology has been directed to the fields such as virus, tumour, pharmacology and food health Journal of Sex Research.Cell cryopreservation and recovery are thin
The fundamental operation of born of the same parents' culture, it is existing to freeze and resuscitation technique there are operating procedures more, frozen stock solution complicated component, big to cellular damage
And the problems such as high, is required to preservation condition, in order to overcome it is existing freeze and method for resuscitation existing for operating procedure more times it is long, from
The heart damages cell and injures the problems such as preservation condition is high, and the invention discloses a kind of easy, efficient cell cryopreservation and recovery sides
Method.
Summary of the invention
The present invention is for the above-mentioned the deficiencies in the prior art of customer service, it is desirable to provide a kind of cell cryopreservation of simplicity and recovery side
Method.Cell cryopreservation and method for resuscitation disclosed in this invention are easy to operate, rationally effectively, when especially solving no liquid nitrogen condition
The problem of cell cryopreservation.
Easy cell cryopreservation and method for resuscitation of the invention, include the following steps:
Step 1: freezing
1a, taking-up is fused to 80% or more cell from incubator, abandons culture medium, is buffered with the PBS for being preheating to 37 DEG C
Liquid cleans cell 1-2 times, abandons whole PBS buffer solution；
1b, trypsase is added (to press 25cm into the Tissue Culture Flask of 1a2Culture bottle in plus 0.5-1mL trypsase
Ratio addition), then vitellophag 5-50s abandons trypsase；
1c, fresh culture is added into the Tissue Culture Flask of 1b, gently blows and beats, prepares cell suspension；
1d, the 1d cell suspension obtained is transferred in centrifuge tube, 1000-1500r/min is centrifuged 3-10min；
Supernatant is abandoned after 1e, centrifugation, adds appropriate cells frozen storing liquid that cell is resuspended, cell suspension is then transferred to cell cryopreservation
Guan Zhong；The cells frozen storing liquid includes the fetal calf serum of 85%-95% and the DMSO of 5%-15%；Cell is resuspended in cells frozen storing liquid
Dosage are as follows: every milliliter of frozen stock solution is resuspended 1 × 106-1×107A cell；
1f, the 1e cryopreservation tube equipped with cell suspension obtained is placed in 4 DEG C of refrigerator 10-30min is pre-chilled, then shifted
1-2h is freezed into -20 DEG C of refrigerator, finally cell cryopreservation tube is transferred in -80 DEG C of ultra low temperature freezer and is saved for a long time.?
It does not influence to save duration up to 9 months under conditions of cell activity.
Step 2: recovery
2a, the cell cryopreservation tube frozen is taken out from -80 DEG C, be immediately placed in the water-bath for being pre-heated to 37-40 DEG C, dissolved
Process is constantly shaken cryopreservation tube to solid and is completely dissolved, to accelerate solution rate；
2b, rapidly by dissolved cell suspension be transferred to equipped with proper amount of fresh culture medium (every milliliter recovery after cell
In suspension be added 3-8mL fresh culture) culture bottle in, be then placed in 37 DEG C, 5%CO2Incubator in cultivate, 24-48h
Most cells are adherent, continue culture to 48-72h, until cell fusion is used or passed for test cell line up to passage after 90% or more
It is frozen after generation.
The fetal calf serum of high glucose medium and 10-20% of the fresh culture containing 80-90%DMEM.
Compared with prior art, its advantages are mainly reflected in for cell cryopreservation disclosed by the invention and the method for recovery:
1, centrifugation step is few.For the cell of growth in vitro, centrifugal process has one to it after being prepared into cell suspension
Fixed damage, the cell especially just recovered is more fragile, and centrifugal process damages it more greatly, in this method after cell recovery directly
It is transferred in culture bottle and cultivates, be not necessarily to centrifugation step；
2, cells frozen storing liquid ingredient is simple, and only the DMSO of the fetal calf serum comprising 85%-95% and 5%-15%, simplifies
Cell cryopreservation work；
3, this method freeze-stored cell reduces the requirement to conditions of cryopreservation, under conditions of no liquid nitrogen, can still carry out normal
Cell cryopreservation and recovery, under conditions of not influencing cell activity save duration up to 9 months, be able to satisfy common laboratory pair
The requirement of cell cryopreservation and recovery.
Detailed description of the invention
Fig. 1 is cell cryopreservation 6 months, after 9 months recovery density, recovery for 24 hours with form when 48h, wherein left side is classified as
Freeze 6 months cell recovery forms, right side is classified as and freezes 9 months cell recovery forms.Cell in Fig. 1 be 50 × it is aobvious
That observes under micro mirror freezes the cellular morphology recovered after a certain period of time, as can be seen from the figure: freezing 6 months cell densities
Greater than the density (density of cell suspension, unrelated with duration is frozen when density depends on freezing) for freezing 9 months, by adherent equal
It can normal proliferative.Illustrate that the cell that 9 months are frozen according to technical solution of the present invention can normally recover.
Specific embodiment
Technical solution of the present invention is further analyzed and described with specific embodiment with reference to the accompanying drawing.
PBS buffer solution preparation method described in following embodiment (1L formula) is as follows: potassium dihydrogen phosphate (KH2PO4):
0.27g, disodium hydrogen phosphate (Na2HPO4): 1.42g, sodium chloride (NaCl): 8g, potassium chloride (KCl) 0.2g add deionized water about
800mL is sufficiently stirred dissolution, concentrated hydrochloric acid tune pH to 7.4, constant volume to 1L to get pH7.4 PBS.
Embodiment 1:
1、25cm2Culture bottle culture people gastric epithelial cell (Ges-1), taken out from incubator when being fused to 90%, abandon
Culture medium is cleaned cell 2 times with the PBS 2mL for being preheating to 37 DEG C, then abandons PBS；
2, add 1mL trypsase into Tissue Culture Flask, be laid flat culture bottle to trypsase and cover entire culture bottle bottom surface,
Trypsase is abandoned after 20s；
3, the fresh culture 5mL for containing 15% fetal calf serum is added into culture bottle, gently blows and beats, it is outstanding to be prepared into cell
Liquid；
4, it shifts whole cell suspensions 1000r/min in centrifuge tube and is centrifuged 8min；
5, supernatant is abandoned after being centrifuged, cells frozen storing liquid is added into centrifuge tube, cell precipitation is resuspended, then turns cell suspension
It moves in cell cryopreservation tube, this cells frozen storing liquid includes 95% fetal calf serum and 5% DMSO；
6, the cryopreservation tube equipped with cell suspension is placed in 5 DEG C of refrigerator and 30min is pre-chilled, be then transferred to -20 DEG C of ice
1h is freezed in case, finally cell cryopreservation tube is transferred in -80 DEG C of ultra low temperature freezer and is saved 6 months；
The recovery of people's gastric epithelial cell (Ges-1):
7, the ultra low temperature freezer taking-up from -80 DEG C freezes 6 months cell cryopreservation tubes, is immediately placed in and has been warmed up to 39 DEG C
Water-bath in, cryopreservation tube is constantly shaken in course of dissolution to being completely dissolved, to accelerate solution rate；
8, dissolved cell suspension is transferred to the 25cm equipped with 5mL fresh culture rapidly2In culture bottle, fresh training
Support the fetal calf serum that base contains 10%；
9, left and right gently shakes up, until culture medium, which is paved with bottom of bottle face, can be put into 37 DEG C, 5%CO2Incubator in cultivate,
Most cells are adherent for 24 hours, continue culture to 48h, cell fusion is more than 80% (recovery cellular morphology is shown on the left of Fig. 1).Left and right
It gently shakes up and refers to that one high and one low shake of the left and right side of culture bottle (to avoid cell aggregation caused by rotation shake, sprawls unevenness
The problems such as even), until culture medium confluent cultures bottom of bottle face can be put into incubator and cultivate.
Embodiment 2:
1,6 orifice plates culture Ges-1 takes out from incubator when being fused to 90% or more, culture medium is abandoned, with being preheating to 37 DEG C
PBS1mL clean cell 1 time, then abandon PBS；
2, it to Kong Zhongjia 0.5mL trypsase, is laid flat 6 orifice plates to trypsase and covers entire culture bottle bottom surface, abandoned after 20s
Trypsase；
3, the fresh culture 3mL for containing 10% fetal calf serum is added into hole, gently blows and beats, is prepared into cell suspension；
4, it shifts whole cell suspensions 1500r/min in centrifuge tube and is centrifuged 5min；
5, supernatant is abandoned after being centrifuged, cells frozen storing liquid is added into centrifuge tube, cell precipitation is resuspended, then turns cell suspension
It moves in cell cryopreservation tube, this cells frozen storing liquid includes 90% fetal calf serum and 10% DMSO；
6, the cryopreservation tube equipped with cell suspension is placed in 5 DEG C of refrigerator and 20min is pre-chilled, be then transferred to -20 DEG C of ice
2h is freezed in case, finally cell cryopreservation tube is transferred in -80 DEG C of ultra low temperature freezer and is saved 9 months；
The recovery of people's gastric epithelial cell (Ges-1):
7, the ultra low temperature freezer taking-up from -80 DEG C freezes 9 months cell cryopreservation tubes, is immediately placed in and has been warmed up to 37 DEG C
Water-bath in, cryopreservation tube is constantly shaken in course of dissolution to being completely dissolved, to accelerate solution rate；
8, dissolved cell suspension is transferred in the 6 orifice plates equipped with 2mL fresh culture rapidly, fresh culture contains
15% fetal calf serum；
9, left and right gently shakes up, until culture medium, which is paved with bottom of bottle face, can be put into 37 DEG C, 5%CO2Incubator in cultivate,
Most cells are adherent for 24 hours, continue culture to 48h, cell fusion is more than 80%.(recovery cellular morphology is shown on the right side of Fig. 1).Left and right
It gently shakes up and refers to that one high and one low shake of the left and right side of culture bottle (to avoid cell aggregation caused by rotation shake, sprawls unevenness
The problems such as even), until culture medium confluent cultures bottom of bottle face can be put into incubator and cultivate.
Claims (6)
1. a kind of cell cryopreservation and method for resuscitation of simplicity, it is characterised in that include the following steps:
Step 1: freezing
1a, taking-up is fused to 80% or more cell from incubator, abandons culture medium, clear with the PBS buffer solution for being preheating to 37 DEG C
It washes cell 1-2 times, abandons whole PBS buffer solution；
1b, add trypsase into the Tissue Culture Flask of 1a, then vitellophag 5-50s abandons trypsase；
1c, fresh culture is added into the Tissue Culture Flask of 1b, gently blows and beats, prepares cell suspension；
1d, the 1d cell suspension obtained is transferred in centrifuge tube, 1000-1500r/min is centrifuged 3-10min；
Supernatant is abandoned after 1e, centrifugation, adds cells frozen storing liquid that cell is resuspended, then cell suspension is transferred in cell cryopreservation tube；
1f, the 1e cryopreservation tube equipped with cell suspension obtained is placed in 4 DEG C of refrigerator 10-30min is pre-chilled, be then transferred to-
1-2h is freezed in 20 DEG C of refrigerator, finally cell cryopreservation tube is transferred in -80 DEG C of ultra low temperature freezer and is saved for a long time；
Step 2: recovery
2a, the cell cryopreservation tube frozen is taken out from -80 DEG C, be immediately placed in the water-bath for being pre-heated to 37-40 DEG C, course of dissolution
It constantly shakes cryopreservation tube to solid to be completely dissolved, to accelerate solution rate；
2b, dissolved cell suspension is transferred in the culture bottle equipped with fresh culture rapidly, is then placed in 37 DEG C, 5%
CO2Incubator in cultivate, 24-48h most cells are adherent, continue culture to 48-72h, until cell fusion is up to 90% or more
After pass on for test cell line use or pass on after freeze.
2. according to the method described in claim 1, it is characterized by:
In step 1b, by 25cm2Culture bottle in plus 0.5-1mL trypsase ratio add trypsase.
3. according to the method described in claim 1, it is characterized by:
In step 1e, the cells frozen storing liquid includes the fetal calf serum of 85%-95% and the DMSO of 5%-15%.
4. method according to claim 1 or 3, it is characterised in that:
The dosage of cells frozen storing liquid resuspension cell are as follows: every milliliter of frozen stock solution is resuspended 1 × 106-1×107A cell.
5. according to the method described in claim 1, it is characterized by:
The fetal calf serum of high glucose medium and 10-20% of the fresh culture containing 80-90%DMEM.
6. according to the method described in claim 1, it is characterized by:
In step 2b, 3-8mL fresh culture is added in the cell suspension after every milliliter of recovery.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107258766A (en) * | 2017-06-18 | 2017-10-20 | 广东博溪生物科技有限公司 | A kind of cell freezing method and cells frozen storing liquid |
| CN111849880A (en) * | 2020-06-23 | 2020-10-30 | 和携科技有限公司 | Recovery method of human adipose mesenchymal stem cells after ultralow-temperature cryopreservation |
| CN112021304A (en) * | 2020-09-17 | 2020-12-04 | 山东省齐鲁干细胞工程有限公司 | Cryopreservation method and recovery method of umbilical cord mesenchymal stem cells |
| CN113293136A (en) * | 2021-05-24 | 2021-08-24 | 成都新生命霍普医学检验实验室有限公司 | Method for reviving cells and application |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107258766A (en) * | 2017-06-18 | 2017-10-20 | 广东博溪生物科技有限公司 | A kind of cell freezing method and cells frozen storing liquid |
| CN111849880A (en) * | 2020-06-23 | 2020-10-30 | 和携科技有限公司 | Recovery method of human adipose mesenchymal stem cells after ultralow-temperature cryopreservation |
| CN111849880B (en) * | 2020-06-23 | 2022-04-29 | 和携科技有限公司 | Recovery method of human adipose mesenchymal stem cells after ultralow-temperature cryopreservation |
| CN112021304A (en) * | 2020-09-17 | 2020-12-04 | 山东省齐鲁干细胞工程有限公司 | Cryopreservation method and recovery method of umbilical cord mesenchymal stem cells |
| CN113293136A (en) * | 2021-05-24 | 2021-08-24 | 成都新生命霍普医学检验实验室有限公司 | Method for reviving cells and application |
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Application publication date: 20190412 |