CN106942202A - A kind of serum-free freezing media for MDBK cells - Google Patents
A kind of serum-free freezing media for MDBK cells Download PDFInfo
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- CN106942202A CN106942202A CN201710284804.2A CN201710284804A CN106942202A CN 106942202 A CN106942202 A CN 106942202A CN 201710284804 A CN201710284804 A CN 201710284804A CN 106942202 A CN106942202 A CN 106942202A
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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
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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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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Abstract
The present invention relates to a kind of serum-free freezing media for MDBK cells, comprising component and concentration of volume percent be：9~11% dimethyl sulfoxide (DMSO), 1~3% Dextran 40 solution, surplus are DMEM high glucose mediums, wherein, Dextran 40 solution is the DMEM high glucose medium solution for the Dextran 40 that mass percent concentration is 10%.The aqueous trehalose that the Dextran 40 solution that concentration of volume percent is 1~3% can also be 6~8% by concentration of volume percent is replaced, and aqueous trehalose is the DMEM high glucose medium solution for the trehalose that concentration is 1mol/L.The freezing media of the present invention is due to the serum without animal origin, it is to avoid the virus pollution of animal origin, and after recovering Cell viability with being remained basically stable compared with adding the culture medium of cow's serum.
Description
Technical field
The invention belongs to cell and technical field of bioengineering, it is related to a kind of serum-free for MDBK cells and freezes culture
Base.
Background technology
Cell culture technology is widely used in the research of life science, is necessary in an experiment to the cold of cell line progress
Freeze the progress for preserving and reserving seed for planting and facilitating follow-up different time experiment.Freezing for successive cell strain can reduce cell because of Secondary Culture
Caused hereditary variation and morphologic change and finite cell lines are avoided aging or vicious transformation occur.Usually use glycerine or two
Methyl sulfoxide (DMSO) and serum addition carry out freeze-stored cell in basal medium, and these additives can protect cell resistance thin
Freezing injury caused by intracellular ice crystal and osmotic effect.Cryoprotector refers to that the thing of freezing injury can be protected cells from
Matter, generally can be divided into permeability and the class of impermeability two.General impermeability protective agent is macromolecule polyalcohol, it is impossible to penetrated into
Cell interior, by diluting the concentration of extracellular electrolyte, reduces solute damage and bound water molecule, reduces containing for extracellular Free water
Amount, reduces ice crystal damage.Conventional has：Sucrose, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), glucan
(dextran), albumin (albumin) and HES (HES) etc..And permeability protective agent is mainly then small molecule
Material, how soluble in water, the ability with water molecules is strong, and easy penetration cell film enters cell interior, can reduce in the cell
The freezing point of cell；And permeability of the cell membrane to water is improved, reduce ice crystal and formed；ECW is promoted to enter thin during recovery
Born of the same parents, alleviate and damaged caused by permeability swelling；The concentration of electrolyte in the solution that do not freeze is diluted, solute damage is reduced.Mainly have
It is several below：Glycerine, dimethyl sulfoxide (DMSO) (DMSO), propane diols, ethylene glycol etc..
At present, the most frequently used technology of cell cryopreservation is liquid nitrogen frozen preservation method, mainly slow using appropriate protective agent is added
Slow freezing carrys out freeze-stored cell, if cell be not added with it is any it is protectant in the case of directly freezed, the moisture content meeting of intraor extracellular
Ice crystal is quickly formed, so as to cause a series of adverse reactions, such as：Cell dehydration makes partial electrolysis matter concentration increase, cause pH value
Change, partially protein is denatured for these reasons, so as to cause cell interior space structure disorderly, thus lysosome membrane meets with
Lysosomal enzyme is discharged to damage, intracellular structure composition is damaged, mitochondrial swelling, function is lost, and causes energy
Measure dysbolism.Class lipoprotein complex on after birth is also easily destroyed the change for causing permeability of cell membrane, makes cell content
Thing is lost.If intracellular ice crystal forms more, with the reduction of cryogenic temperature, ice crystal volumetric expansion causes nucleus DNA space
Irreversible damage occurs for configuration, and causes cell death.Cell cryopreservation and the basic principle of recovery are that slow freeze is melted soon, so can be with
Cell viability is preserved to greatest extent.The method that freeze-stored cell is mixed using serum with DMSO in the prior art, because of animal
Individual different, the serum place of production, lot number are different, and very big per quality of lot difference, its composition can not be consistent；It may be brought into materials
Mycoplasma, virus, produce potential impact to cell, may cause the failure of an experiment or experimental result unreliability, and serum cost
It is high.
Nineteen fifty-seven 2 month 18, S.H. Madin and N.B. Darby is established from an apparent normal adult Ren Bovis seu Bubali
MDBK cell lines.Usual MDBK cells are the epithelioid cells grown with adherent manner.At present, MDBK cell lines are widely used in
The propagation of a variety of ox source viruses and purifying, such as：The type of bovine parainfluenza virus 3 (Bovine parainfluenza virus
Type3, BPIV3), bovine viral diarrhea virus（Bovine Viral Diarrhea Virus ,BVDV）, bovine herpes virus
1 type (Bovine herpesvirus 1, BoHV-1), Bovine Respiratory Syncytial virus (bovine respiratory
Syncytial virus, BRSV) etc..It is existing to freeze scheme frequently with 10%DMSO and 20 ~ 50% for MDBK cells
The FBS scheme that freezes goes to preserve MDBK cells, and the program can be good at as cells with nutrient material, and this contains serum cell jelly
Deposit scheme its cell recoveries and about can reach 85 ~ 90%, Cell viability about can reach 93 ~ 95% or so.Cell freezes through the short time
Deposit the motility rate for remaining to maintain higher again after recovering.But, the hyclone or calf serum used in cell culture and in freezing
Some problems, especially MDBK cells can be caused as ox source cell system, be widely used in research and the vaccine system of ox source virus
Standby to wait research, it is sensitive to the virus existed in cow's serum, and easily it is caused applied to cell culture and the Serology Quality frozen
Influence.And in recent years, the low serum of many animal cell lines being directed to including MDBK cells, serum free medium and
The research of this kind of training method has become focus, promotes us to need the mode that exploitation serum-free freezes to remain this without blood
Clear training mode.
The content of the invention
It is an object of the invention to provide a kind of serum-free freezing media for MDBK cells, for solving freezing at present
, it is necessary to add hyclone or calf serum when depositing MDBK cells, the problem of ox source is viral is readily incorporated.
The present invention is achieved through the following technical solutions：A kind of serum-free freezing media for MDBK cells, comprising
Component and concentration of volume percent be：9~11% dimethyl sulfoxide (DMSO), 1~3% Dextran 40 solution, surplus is DMEM
High glucose medium, wherein, described Dextran 40 solution is that the DMEM for the Dextran 40 that mass percent concentration is 10% is high
Sugar culture-medium solution.The present invention originates without special limitation to the DMEM high glucose mediums, specifically, can use
The specification that Gibco companies provide is 500mL/ bottles of DMEN high glucose mediums, and the culture medium is conventional commercially available prod.
Further, the concentration of volume percent of described dimethyl sulfoxide (DMSO) is 10%.The present invention is to the dimethyl sulfoxide (DMSO)
Source without special limitation, using the commercial goods of routine.
Further, the concentration of volume percent of described Dextran 40 solution is 1%.The present invention is to the dextrose
The source of acid anhydride 40 is without special limitation, specifically, can be carried using Hefei Co., Ltd of Bo Mei biotechnologies Co., Ltd
The Dextran 40 powder of confession.
As a further improvement on the present invention, described concentration of volume percent is 1~3% Dextran 40 solution quilt
Concentration of volume percent is replaced for 6~8% aqueous trehalose, and described aqueous trehalose is the trehalose that concentration is 1mol/L
DMEM high glucose medium solution.
Further, the concentration of volume percent of described dimethyl sulfoxide (DMSO) is 10%.The present invention is to the dimethyl sulfoxide (DMSO)
Source without special limitation, using the commercial goods of routine.
Further, the concentration of volume percent of described aqueous trehalose is 7%.The present invention comes to the trehalose
Source does not have special limitation, specifically, the marine alga that can be provided using Hefei Co., Ltd of Bo Mei biotechnologies Co., Ltd
Icing Sugar end.
Specifically, cell concentration must not be less than 4~5 × 10 in freezing media6cells/ml。
Using the good effect of above-mentioned technical proposal：The easy penetration cells of DMSO in the present invention, during cell cryopreservation
Freeze point depression can be made, the intracellular chance for forming ice crystal is reduced, so as to reduce damage of the ice crystal to cell；Dextran 40 and
Trehalose then plays a part of stabilizing cell membrane, it is to avoid cell is by freezing injury；DMEM high glucose mediums then maintain electrolyte
Balance and offer nutritional ingredient；Due to the serum without animal origin, it is to avoid the virus pollution of animal origin, experimental result
Show, using cell recovery rate of the two kinds of technical schemes of frozen stock solution in the present invention after 90d is frozen be respectively 92.77 ±
2.99% and 92.74 ± 1.23%.
Brief description of the drawings
Fig. 1 is that the cell state for cultivating 12h after MDBK cells 90d after recovery is frozen using technical scheme one
Figure；
Fig. 2 is that the cell state figure for cultivating 12h after MDBK cells 90d after recovery is frozen using technical scheme two；
Fig. 3 is to freeze the cell state figure that control group freezes culture 12h after recovery after MDBK cells 90d using serum.
Embodiment
The source of biomaterial in the present invention：
1st, MDBK cells are purchased from China Veterinery Drug Inspection Office.
Technical scheme is described further with reference to specific embodiment and test example, but should not be managed
Solve as limitation of the present invention：
Embodiment 1
A kind of serum-free freezing media for MDBK cells, comprising component and concentration of volume percent be：11% diformazan
Base sulfoxide, 2% Dextran 40 solution, surplus are DMEM high glucose mediums.Wherein, described Dextran 40 solution is matter
Measure percent concentration for 10% Dextran 40 DMEM high glucose medium solution, refer to using DMEM high glucose mediums as
Solvent, the solution that mass percent concentration is 10% is diluted to by Dextran 40.
Embodiment 2
A kind of serum-free freezing media for MDBK cells, comprising component and concentration of volume percent be：10% diformazan
Base sulfoxide, 1% Dextran 40 solution, surplus are DMEM high glucose mediums.Wherein, described Dextran 40 solution is matter
Measure percent concentration for 10% Dextran 40 DMEM high glucose medium solution, refer to using DMEM high glucose mediums as
Solvent, the solution that mass percent concentration is 10% is diluted to by Dextran 40.
Embodiment 3
A kind of serum-free freezing media for MDBK cells, comprising component and concentration of volume percent be：9% diformazan
Base sulfoxide, 3% Dextran 40 solution, surplus are DMEM high glucose mediums.Wherein, described Dextran 40 solution is matter
Measure percent concentration for 10% Dextran 40 DMEM high glucose medium solution, refer to using DMEM high glucose mediums as
Solvent, the solution that mass percent concentration is 10% is diluted to by Dextran 40.
Embodiment 4
A kind of serum-free freezing media for MDBK cells, comprising component and concentration of volume percent be：9% diformazan
Base sulfoxide, 8% aqueous trehalose, surplus are DMEM high glucose mediums.Wherein, described aqueous trehalose is that concentration is 1mol/
The DMEM high glucose medium solution of L trehalose, refers to DMEM high glucose mediums as solvent, trehalose is diluted to dense
Spend the solution for 1mol/L.
Embodiment 5
A kind of serum-free freezing media for MDBK cells, comprising component and concentration of volume percent be：10% diformazan
Base sulfoxide, 7% aqueous trehalose, surplus are DMEM high glucose mediums.Wherein, described aqueous trehalose is that concentration is 1mol/
The DMEM high glucose medium solution of L trehalose, refers to DMEM high glucose mediums as solvent, trehalose is diluted to dense
Spend the solution for 1mol/L.
Embodiment 6
A kind of serum-free freezing media for MDBK cells, comprising component and concentration of volume percent be：11% diformazan
Base sulfoxide, 6% aqueous trehalose, surplus are DMEM high glucose mediums.Wherein, described aqueous trehalose is that concentration is 1mol/
The DMEM high glucose medium solution of L trehalose, refers to DMEM high glucose mediums as solvent, trehalose is diluted to dense
Spend the solution for 1mol/L.
Test example 1
Serum-free freezes MDBK cells：
Using the adherent MDBK cell surfaces of PBS twice, it is as gentle as possible during separation using trypsin, make
Damage to cell is reduced to minimum.In DMEM culture mediums, suspend separation cell again, determines vibrant cell number.With big
About 200r/min centrifuges 5 minutes sedimentation cells.Supernatant is removed to minimum volume using pipette, should not confuse cell.
Freezing media is made using embodiment 1-3 technical scheme.
Each cryopreservation tube frozen stock solution adds 1.5ml freezing media, initial freeze-stored cell number for average 3.8 ×
106Cells/ml, Cell viability 99.1%.The suspension cell in frozen stock solution, is dispensed into cryopreservation tube, is set up three groups of repetitions, will be frozen
Deposit pipe and be put into 90min in 30min in 4 DEG C of refrigerators, -20 DEG C of refrigerators, cryopreservation tube is being put into -80 DEG C of refrigerator overnight, then
It is transferred in liquid nitrogen and stores.
Freeze the recovery of MDBK cells.Freeze-stored cell is more fragile, gentle manipulation.Freeze-stored cell wants fast melt, and directly
Connect in the complete growth medium of addition.Cell is sensitive to freezing agent DMSO, and centrifugation removes freezing media, then adds completely raw
In long culture medium.Stored cells are taken out, the side recovered again in 37 DEG C of thermostat water baths after first dissolving at 40 DEG C is selected in this experiment
Formula carries out cell recovery, frozen stock solution temperature and not up to 40 DEG C of bath temperature where cell, but accelerate simultaneously freeze it is thin
The thawing time of born of the same parents, is conducive to rapid fluid resuscitation, improves recovery survival rate.1.5ml freeze-stored cells are added to about 15ml complete
Growth medium, is gently mixed.Centrifuged 5 to 10 minutes with about 1000r/min, supernatant discarded, with 10%FBS culture mediums gently
Pressure-vaccum makes cell suspend again, is transferred in 25cm2 square vases and cultivates.Finally, it is put into 5% CO2 humidity, 37 DEG C of constant incubators
Culture.
After taking after the trypan blues of 10 μ L 0.4% and 10 μ L freeze-stored cells are recovered that mixed liquor is uniform and mixing, liquid-transfering gun point sample is used
To blood counting chamber, it is placed under inverted microscope and observes, counts.Microscope photograph is as shown in Figure 1.
Cell counts are as follows after technical scheme one is recovered, total cell number (1.66 ± 0.31) × 10 after recovery6, cell
The rate of recovery is 47.42 ± 8.86%, and viable count is (1.54 ± 0.24) × 106, Cell viability is 92.77 ± 2.99%.
Test example 2
Serum-free freezes MDBK cells：
Using the adherent MDBK cell surfaces of PBS twice, it is as gentle as possible during separation using trypsin, make
Damage to cell is reduced to minimum.In DMEM culture mediums, suspend separation cell again, determines vibrant cell number.With big
About 200r/min centrifuges 5 minutes sedimentation cells.Supernatant is removed to minimum volume using pipette, should not confuse cell.
Freezing media is made using embodiment 4-6 technical scheme.
Each cryopreservation tube frozen stock solution adds 1.5ml freezing media, initial freeze-stored cell number for average 3.8 ×
106Cells/ml, Cell viability 99.1%.The suspension cell in frozen stock solution, is dispensed into cryopreservation tube, is set up three groups of repetitions, will be frozen
Deposit pipe and be put into 90min in 30min in 4 DEG C of refrigerators, -20 DEG C of refrigerators, cryopreservation tube is being put into -80 DEG C of refrigerator overnight, then
It is transferred in liquid nitrogen and stores.
Freeze the recovery of MDBK cells.Freeze-stored cell is more fragile, gentle manipulation.Freeze-stored cell wants fast melt, and directly
Connect in the complete growth medium of addition.Cell is sensitive to freezing agent DMSO, and centrifugation removes freezing media, then adds completely raw
In long culture medium.Stored cells are taken out, the side recovered again in 37 DEG C of thermostat water baths after first dissolving at 40 DEG C is selected in this experiment
Formula carries out cell recovery, frozen stock solution temperature and not up to 40 DEG C of bath temperature where cell, but accelerate simultaneously freeze it is thin
The thawing time of born of the same parents, is conducive to rapid fluid resuscitation, improves recovery survival rate.1.5ml freeze-stored cells are added to about 15ml complete
Growth medium, is gently mixed.Centrifuged 5 to 10 minutes with about 1000r/min, supernatant discarded, with 10%FBS culture mediums gently
Pressure-vaccum makes cell suspend again, is transferred in 25cm2 square vases and cultivates.Finally, it is put into 5% CO2 humidity, 37 DEG C of constant incubators
Culture.
After taking after 10 μ L0.4% trypan blues and 10 μ L freeze-stored cells are recovered that mixed liquor is uniform and mixing, arrived with liquid-transfering gun point sample
Blood counting chamber, is placed under inverted microscope and observes, counts (being repeated 3 times counting).Microscope photograph is as shown in Figure 2.
Cell counts are as follows after technical scheme two is recovered, total cell number (1.24 ± 0.21) × 106, cell after recovery
The rate of recovery is 35.43 ± 6.00%, and viable count is (1.15 ± 0.18) × 106, and Cell viability is 92.74 ± 1.23%.
Test example 3
Operating method uses total volume fraction 10%DMSO and 20%FBS and surplus for the high sugar cultures of DMEM with test example 1-2
Base freeze scheme go preserve MDBK cells, serum freeze control group recovery after cell counts it is as follows, total cell after recovery
Number is（5.27±0.34)×106, cell recoveries are 90.83 ± 0.05%, and viable count is（5.05±0.18)×106, carefully
Born of the same parents' motility rate is 95.82 ± 3.41%.Microscope photograph is as shown in Figure 3.
Cell recovery of the serum-free freezing media of the present invention after 90d is frozen it can be seen from test example 1-3 contrasts
Rate is compared with the culture medium of conventional addition cow's serum, and Cell viability is almost suitable after recovery, and due to without animal origin
Serum, it is to avoid the virus pollution of animal origin, it is ensured that the progress of later experiments and the reliability of guarantee experimental result.
Claims (7)
1. a kind of serum-free freezing media for MDBK cells, it is characterised in that：Comprising component and percent by volume it is dense
Spend and be：9~11% dimethyl sulfoxide (DMSO), 1~3% Dextran 40 solution, surplus are DMEM high glucose mediums, wherein, it is described
Dextran 40 solution be the Dextran 40 that mass percent concentration is 10% DMEM high glucose medium solution.
2. cells frozen storing liquid according to claim 1, it is characterised in that：The percent by volume of described dimethyl sulfoxide (DMSO) is dense
Spend for 10%.
3. cells frozen storing liquid according to claim 1, it is characterised in that：The volume basis of described Dextran 40 solution
Specific concentration is 1%.
4. cells frozen storing liquid according to claim 1, it is characterised in that：Described concentration of volume percent is 1~3%
Dextran 40 solution is replaced by concentration of volume percent for 6~8% aqueous trehalose, and described aqueous trehalose is concentration
For the DMEM high glucose medium solution of 1mol/L trehalose.
5. cells frozen storing liquid according to claim 4, it is characterised in that：The percent by volume of described dimethyl sulfoxide (DMSO) is dense
Spend for 10%.
6. cells frozen storing liquid according to claim 4, it is characterised in that：The percent by volume of described aqueous trehalose is dense
Spend for 7%.
7. the MDBK cells according to claim 1 or 4, it is characterised in that：Cell concentration must not be less than 4 in freezing media
~5 × 106cells/ml。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109221089A (en) * | 2018-10-16 | 2019-01-18 | 浙江美泰生物科技有限公司 | A kind of method that the frozen stock solution and adipose tissue of adipose tissue freeze |
| CN113519506A (en) * | 2021-09-07 | 2021-10-22 | 依科赛生物科技（太仓）有限公司 | Protein-free and DMSO-free cell cryopreservation liquid, application and preparation method thereof |
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| CN104719282A (en) * | 2015-02-13 | 2015-06-24 | 广州赛莱拉干细胞科技股份有限公司 | Peripheral blood mononuclear cell serum-free freezing medium and freezing method |
| CN105532650A (en) * | 2016-03-10 | 2016-05-04 | 广州赛莱拉干细胞科技股份有限公司 | Cell cryopreservation solution |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102763639A (en) * | 2012-07-19 | 2012-11-07 | 童亚林 | Composite anti-freezing liquid, and method for preserving human amnion by using same |
| CN104719282A (en) * | 2015-02-13 | 2015-06-24 | 广州赛莱拉干细胞科技股份有限公司 | Peripheral blood mononuclear cell serum-free freezing medium and freezing method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109221089A (en) * | 2018-10-16 | 2019-01-18 | 浙江美泰生物科技有限公司 | A kind of method that the frozen stock solution and adipose tissue of adipose tissue freeze |
| CN113519506A (en) * | 2021-09-07 | 2021-10-22 | 依科赛生物科技（太仓）有限公司 | Protein-free and DMSO-free cell cryopreservation liquid, application and preparation method thereof |
| CN113519506B (en) * | 2021-09-07 | 2021-12-31 | 依科赛生物科技（太仓）有限公司 | Protein-free and DMSO-free cell cryopreservation liquid, application and preparation method thereof |
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