CN106982820A - Compound cryosar freezes the method that system freezes schwann cell - Google Patents
Compound cryosar freezes the method that system freezes schwann cell Download PDFInfo
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- CN106982820A CN106982820A CN201710266580.2A CN201710266580A CN106982820A CN 106982820 A CN106982820 A CN 106982820A CN 201710266580 A CN201710266580 A CN 201710266580A CN 106982820 A CN106982820 A CN 106982820A
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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 method that system freezes schwann cell is frozen the invention discloses a kind of compound cryosar.Comprise the following steps：By amino acids gelator heating be dissolved in cell culture medium, filtering, it is degerming, prepare cell suspending liquid with the cell culture medium containing gelator；It is added dropwise at 04 DEG C into the cell suspending liquid and contains the protectant cell culture medium of composite frozen, is well mixed；The composite frozen protective agent is the mixing by permeability cryoprotector and impermeability cryoprotector；Cryopreservation tube is put into program freezing storing box to freeze after 24h at 80 DEG C, then will be frozen in cryopreservation tube immigration liquid nitrogen.The supermolecular gel that amino acids gelator is self-assembly of has unique tridimensional network, and when cryoprotector is added dropwise, cell is wrapped up by supermolecular gel, reduces the osmotic injury of cryoprotector；During cooling and rewarming, because the tridimensional network of supermolecular gel limits growth and the recrystallization of ice crystal, so as to reduce ice crystal damage.
Description
Technical field
The invention belongs to cell cryopreservation technical field, and in particular to a kind of method of freezing schwann cell.
Background technology
The reparation and regeneration of nerve always biology and the study hotspot of organizational project, and schwann cell
(Schwanncell) played an important role in terms of the regeneration of nerve.Therefore many related researchs are required for using
Schwann cell, then how external substantial amounts of storage schwann cell is just into a great problem, and cryopreservation is most common at present
And effective store method.
Cryopreservation is that cell is placed in ultralow temperature (being usually -80 DEG C or lower) environment, suppresses the new old of cell
It is metabolized to reach the purpose of long term storage.At present, to be successfully applied to stem cell, erythrocyte, liver thin for cryopreservation technology
During the preservation of the cells such as born of the same parents, sperm, egg mother cell, bone cellses, skin, cornea and tissue.Low-temperature frozen on cell
Deposit and have pertinent literature report both at home and abroad.The method of cryopreservation mainly has two kinds of sequencing slow cryopreservation and glass frozen preservation,
Damage in refrigerating process is mainly caused by the factor of following two：(1) solution is damaged：In temperature-fall period in Extracellular solution
Water is first crystallized so that extracellular fluid osmotic pressure is increased, and intracellular moisture can be penetrated into largely in Extracellular solution, cause cell
Dehydration is drastically shunk, and causes cellular damage even dead；(2) intracellular ice crystal is damaged：Equally in temperature-fall period, when temperature reaches
To intracellular fluid freezing point when, the water in intracellular fluid can start crystallization, and inevitably damage is caused to cell, and freeze and protect
Shield agent can reduce intracellular water content and play a part of protecting cell.It is directed to optimizing the research of freezing scheme at this stage,
Search out a kind of cryopreservation method safely, effectively, easy.
Cryoprotector refers to the material from damage that cell or tissue is protected in cell or tissue cryopreservation.Freezing
Protective agent can be divided into two kinds of permeability cryoprotector and impermeability cryoprotector according to the difference of protection mechanism.Infiltration
Property cryoprotector (Permeable Cryoprotectants, PC) is also known as " intracellular cryoprotector ", and it can be penetrated into carefully
Intracellular portion, usually small-molecule substance, it is readily permeable to enter intracellular, intracellular most of water is cemented out, reduction is thin
The electrolyte concentration of extracellular solution, reduces intraor extracellular osmotic pressure, it is to avoid cell dehydration is excessive, while it can also make cell
Internal solution reaches the concentration required for vitrifying.Conventional permeability cryoprotector has dimethyl sulfoxide (DMSO), ethylene glycol, the third two
Alcohol, glycerine etc..Impermeability cryoprotector (Impermeable Cryoprotectants, IPC) is also known as that " extracellular fluid is cold
Freeze protective agent ", this cryoprotector is mainly macromolecular substances, and it is impermeable to cell interior, but extracellular
Its osmotic pressure is improved in solution, freezing point is reduced, the formation of ice crystal is reduced, so as to reach the purpose of protection cell or tissue.It is common
Impermeability cryoprotector have macromolecular substances and the carbohydrates such as sucrose, trehalose, glucose, poly- diethanol.Impermeability
Cryoprotector is usually that biocompatibility does not have cytotoxic material well, therefore generally by itself and permeability cryoprotection
Agent collective effect, on the one hand can reduce the consumption of permeability cryoprotector, on the other hand can improve cell and tissue
Protecting effect.
Supermolecular gel is a kind of physical crosslinking gel, it be passed through by a small amount of low molecule gelator it is intermolecular non-
What covalent effect was self-assembly of in medium (water or organic solvent).Therefore supermolecular gel typically has well degradable
Property and thixotropy, and be reversible；Because the activation energy of non-covalent bond is relatively low, after the stimulation by external environment, surpass
Molecular gel easily makes corresponding response.The supermolecular gel such as formed by hydrogen bond action has temperature-responsive；Pass through
The supermolecular gel of electrostatic interaction formation is to pH and electrolyte concentration sensitivity etc..
Supermolecular gel can be used for preparing the fields such as composite, biology sensor, medicine carrying material.But it is rarely seen by supermolecule
Gel rubber system is used for the report of the cryopreservation of cell.
The content of the invention
Present invention aims at provide a kind of method that utilization NEW TYPE OF COMPOSITE cryopreservation system freezes schwann cell, the body
System can reduce the damage that schwann cell is subject to during freezing, and improve the survival rate after its recovery and maintain cell function, from
And play effective cryoprotection effect.
It is as follows using technical scheme to reach above-mentioned purpose：
Compound cryosar freezes the method that system freezes schwann cell, comprises the following steps：
1) heating of amino acids gelator is dissolved in cell culture medium, it is filtering, degerming, with containing the thin of gelator
Born of the same parents' culture medium prepares cell suspending liquid；
2) supermolecular gel is formed at 0-4 DEG C；
3) it is added dropwise and contains the protectant cell culture medium of composite frozen, the system for the treatment of reaches swelling equilibrium；The composite frozen
Protective agent is the mixing by permeability cryoprotector and impermeability cryoprotector；
4) it is put into cryopreservation tube to freeze in program freezing storing box, then will be frozen in cryopreservation tube immigration liquid nitrogen.
By such scheme, the amino acids gelator is prepared as follows：
Dodecyl-Boc-L- methyl-P-tyrosines are prepared by alkylated reaction with Boc-L- methyl-P-tyrosines
(BDTE)；
Dodecyl-Boc-L- methyl-P-tyrosines prepare dodecyl-Boc-L- tyrosine by hydrolysis
(BDT), it is described amino acids gelator.
By such scheme, described cell culture medium be add in RPMI1640 culture mediums 10vol% hyclones and
The full culture medium of cell being prepared into after 1vol% is dual anti-.
By such scheme, described permeability cryoprotector is dimethyl sulfoxide (DMSO), and impermeability cryoprotector is sea
Algae sugar, the two final concentration is respectively 7.5~8.5vol% and 0.045~0.055mol/L.
By such scheme, step 3 is first added dropwise the cell culture medium containing impermeability cryoprotector, then is added dropwise containing oozing
The cell culture medium of permeability cryoprotector.
Apply to K-K models when freezing protective agent is added during cell cryopreservation, there are two basic assumptions：(1)
Intracellular fluid is ideal dilute solution；(2) cell membrane is uniform pellicle.K-K models can be represented with below equation：
Wherein, VcRepresent cell volume, LpRepresent cell membrane for the infiltration coefficient of water, PsRepresent cell membrane for freezing
Protectant infiltration coefficient, AcThe surface area of cell membrane is represented, R represents ideal gas constant, and T represents absolute temperature, NsRepresent thin
The osmolality of intracellular solution,Represent the average osmolality of intraor extracellular solution, MnRepresent that the same of impermeable liquid oozes weight
Rub, σ represents reflectance factor；Subscript n represents solute, and s represents freezing protective agent, and subscript i represents intracellular, and e represents extracellular.
Because the supermolecular gel that amino acids gelator is self-assembly of generally has unique tridimensional network,
When cryoprotector is added dropwise, gel is swelled, with the dropwise addition of cryoprotector, reaches swelling equilibrium, now cell quilt
Supermolecular gel is wrapped up, and is reduced cell membrane to water and the infiltration coefficient of cryoprotector, can effectively be slowed down cell body
Long-pending change, slows down the acute variation of intraor extracellular osmotic pressure so that permeability cryoprotector DMSO infiltration rates are reduced, and are subtracted
The small osmotic injury of cryoprotector, and due to the presence of impermeability cryoprotector trehalose so that extracellular fluid oozes
Pressure is improved thoroughly, reduces freezing point, reduces the formation of ice crystal, and trehalose can also simultaneously reduce in cell appearance into layer protecting film
Osmotic injury and the ice crystal damage that cell is subject to；During cooling and rewarming, because supermolecular gel system is spatially
Limitation, the growth of ice crystal and recrystallization be inhibited, so as to reduce ice crystal damage.
Being used in conjunction with for permeability and impermeability cryoprotector, on the one hand can reduce poisonous permeability freezing and protect
Agent DMSO consumption is protected, the cryoprotective effect of cell on the other hand can be improved.
The cell culture medium containing impermeability cryoprotector is first added dropwise, then is added dropwise containing permeability cryoprotector
Cell culture medium.It is added dropwise after impermeability cryoprotector, on the one hand adds the osmotic pressure of Extracellular solution, it is on the other hand non-
Permeability cryoprotector forms layer protecting film extracellular, and cell is protected jointly with gel；Permeability freezing is being added dropwise
During protective agent, because extracellular fluid osmotic pressure is increased, it is more favorable for cryoprotector and penetrates into cell, simultaneously because there is gel
The double shielding film formed with impermeability cryoprotector, greatly reduces the infiltration that permeability cryoprotector is caused
Damage.
The supermolecular gel that amino acids gelator is self-assembly of has thermal reversibility, therefore during rewarming,
When temperature is reached more than phase transition temperature, supermolecular gel is changed into liquid from gel state, by centrifugation can and cell separation,
The propagation of cell and differentiation after rewarming are not interfered with while operation is not increased.
The present invention compared with prior art, with advantages below：
The supermolecular gel that amino acids gelator is self-assembly of is a kind of physical crosslinking gel, and it is by a small amount of
Low molecule gelator is self-assembly of by intermolecular noncovalent interaction in medium (water, organic solvent).Therefore it is super
Molecular gel typically has good degradability and thixotropy, and is reversible；Due to non-covalent bond activation energy relatively
Low, after the stimulation by external environment, supermolecular gel easily makes corresponding response.Amino acid used in simultaneously of the invention
The gellike factor has good biocompatibility.
Due to the tridimensional network for the supermolecular gel uniqueness that amino acids gelator is self-assembly of, containing solidifying
Cryoprotector is added dropwise after the cell suspending liquid formation gel of the glue factor, cell is wrapped up by supermolecular gel so that freezing is protected
The reduction of agent infiltration rate is protected, the acute variation of the change intraor extracellular osmotic pressure of cell volume is slow down, reduces cryoprotection
The osmotic injury of agent, due to the presence of impermeability cryoprotector, while reducing osmotic injury and ice crystal damage；In cooling
During rewarming, because the limitation of gel rubber system spatially, the growth of ice crystal and recrystallization are inhibited, so as to reduce
Ice crystal damage；Supermolecular gel has thermal reversibility, therefore during rewarming, when temperature is reached more than phase transition temperature,
Supermolecular gel is changed into liquid from gel state, by centrifugation can and cell separation, will not shadow while operation is not increased
Ring the propagation of cell and differentiation after rewarming.
Use permeability and impermeability cryoprotector, wherein impermeable simultaneously on the basis of supermolecular gel system
Property cryoprotector is usually that biocompatibility does not have cytotoxic material well, therefore by itself and permeability cryoprotector
Collective effect, on the one hand can reduce the consumption of permeability cryoprotector, on the other hand can improve the protecting effect of cell,
So the combination of two kinds of cryoprotectors can play more preferable protecting effect during cryopreservation.
Brief description of the drawings
Fig. 1：Microscopic appearance figure under supermolecular gel system room temperature (25 DEG C) on slide；
Fig. 2：The phase transition temperature of supermolecular gel system and the relation of gelator concentration；
Fig. 3：Compound cryosar freezes the microscopic appearance figure on slide in system ice-water bath (0~4 DEG C)；
Fig. 4：Compound cryosar freezes the DSC test results of system；
Fig. 5：The survival rate contrast of control group and experimental group cell；
Fig. 6：MTT testing results.
Embodiment
The present invention is described in detail with reference to embodiment and accompanying drawing.The following examples are simply to present invention work
Further explanation explanation, but it is not exhaustive, any limitation is not done to the present invention.
Embodiment 1
The preparation of supermolecular gel and its performance：
1st, gelator BDT is prepared：
(1) synthesis (alkylation of Boc-L- methyl-P-tyrosines) of dodecyl-Boc-L- methyl-P-tyrosines (BDTE)：
Weigh 3.998g (0.0135mol) Boc-L- methyl-P-tyrosines to be placed in 25mL round-bottomed flasks, add 10ml DMF,
After raw material is completely dissolved, 3.7113g K are added2CO3(0.027mol) is rear to add 4mL bromododecanes as acid binding agent
(0.0167mol), reacts at room temperature 12h.Reaction is obtained after adding 20mL water washings, suction filtration, washing filter cake, vacuum drying 24h after terminating
To white solid.Absolute ethyl alcohol (20ml) is recrystallized 1 time, obtains pure white solid.
(2) BDT synthesis (BDTE hydrolysis)：
0.5007g (0.0011mol) BDTE is placed in 25mL flasks and dissolved with 10mL ethanol, is added dropwise under ice-water bath
Enter 1mol/L NaOH solution 1.7mL (0.0017mol), 1h is reacted at 0 DEG C or so, react at room temperature 5h.Under vigorous stirring to
It is 1~2 that 0.2mol/L sour (25ml) solution of cryosel is added dropwise in reaction system to pH, and suction filtration, vacuum drying 24h is obtained
0.4858g white solids.
The gelator can be self-assembly of the supermolecular gel system with tridimensional network, and cell is wrapped by it
Wrap up in so that cryoprotector infiltration rate is reduced, and reduces the osmotic injury of cryoprotector；During cooling and rewarming,
The growth of ice crystal and recrystallization are inhibited, so as to reduce ice crystal damage.
2. solution needed for supermolecular gel morphology characterization：5mg BDT are added in 1ml RPMI1640 culture mediums, are placed in
Dissolved in 60 DEG C of water-baths, after it is completely dissolved, be cooled to room temperature, dropped on slide, be placed under light microscope and see
Examine microscopic appearance, Fig. 1 is the microscopic appearance figure on slide under supermolecular gel system room temperature (25 DEG C), it can be seen that solidifying
The glue factor is self-assembly of the supermolecular gel with tridimensional network in cell culture fluid RPMI1640, and the structure can be with
Cell is wrapped up in refrigerating process, plays a part of reducing ice crystal damage.
3. taking a certain amount of BDT to be added in 1ml RPMI1640 culture mediums, it is placed in 60 DEG C of water-baths and dissolves, be respectively configured
Into 1g/L, 1.5g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 8g/L BDT culture medium solutions, and determine with falling ball method its phase
Transition temperature, test result is as shown in Figure 2.BDT concentration phase transition temperature in 1g/L to 5g/L is sharply increased, and is reached in concentration
After 5g/L, increase tends towards stability.With the increase of gelator concentration, fiber number increase, the chance mutually wound increases, shape
Into finer and close tridimensional network so that the heat endurance enhancing of supermolecular gel system, so that supermolecular gel body
The phase transition temperature of system increases therewith.
Embodiment 2
Compound cryosar freezes preparation and its performance of system：
The gained BDT of Example 1 is added in 1ml RPMI1640 culture mediums, is placed in 60 DEG C of water-baths and is dissolved, treats that its is complete
It is added dropwise a certain amount of containing the protectant RPMI1640 culture medium solutions of composite frozen after fully dissolved, is prepared into compound cryosar and freezes body
It is stand-by.The ultimate density of the compound system of configuration is 1.5g/LBDT, 8vol%DMSO, 0.05mol/L trehalose, and in ice
Using its microscopic appearance of observation by light microscope in water-bath (0~4 DEG C), as shown in Figure 3.Gelator is protected containing composite frozen
The microstructure of the supermolecular gel formed in the RPMI1640 culture medium solutions for protecting agent is different, but still presents three-dimensional netted
Structure, when permeability cryoprotector permeates, because cell is wrapped by supermolecular gel, the seepage velocity of permeation protective agent
It can substantially reduce, so that the damage that the permeability cryoprotector reduced is caused to cell.
Take the gained BDT of 3mg embodiments 1 to be added in 1ml RPMI1640 culture mediums, be placed in 60 DEG C of water-baths and dissolve, treat it
Isometric (1ml) is added dropwise after being completely dissolved respectively containing 16vol%DMSO and 16vol%DMSO, 0.1mol/L trehalose
RPMI1640 culture medium solutions, are prepared into ultimate density for 1.5g/LBDT, 8vol%DMSO and 1.5g/LBDT, 8vol%
Two kinds of cryopreservation systems of DMSO, 0.05mol/L trehalose, for DSC tests.
Test result such as Fig. 4, shows 1.5g/LBDT, 8vol%DMSO cryopreservation system and 1.5g/L BDT, 8vol%
The freezing point of DMSO, 0.05mol/L trehalose cryopreservation system is respectively -18.9 DEG C and -19.8 DEG C, illustrates the addition of trehalose
The freezing point of whole system can be reduced.
Embodiment 3
The refrigerating process of schwann cell：
1. the configuration of cell culture medium system：The configuration of cell culture medium system：In RPMI1640 culture mediums, add
10vol% hyclones and 1vol% are dual anti-, are prepared into the full culture medium of cell.Full culture medium is divided into two parts, portion and added
Enter the gained BDT gelators (concentration is 1.5g/L) of embodiment 1 to dissolve at 60 DEG C, the filtration sterilization after it is completely dissolved, separately
It is a without any processing, be placed in after packing respectively 4 DEG C it is standby.
2. the configuration of cryoprotector system：Be separately added into full culture medium 16vol%DMSO, 32vol%DMSO,
0.2mol/L trehaloses, 0.22 μm of membrane filtration is degerming, be placed in after packing respectively 4 DEG C it is standby.
3. the refrigerating process of schwann cell：By 0.25% Trypsin Induced of the cell in logarithmic phase, it is divided into three groups
Supernatant is removed after 1000r/m centrifugations 8min, control group dispels cell with full culture medium, takes 0.5ml cell suspending liquids to be placed in 2ml and freezes
Guan Zhong, the balance 5min in ice-water bath (4 DEG C), is added dropwise containing for isometric (0.5ml) after cell suspending liquid is changed into gel state
16vol%DMSO full culture medium, often be added dropwise one or two drip cryopreservation tube need to rock it is several under DMSO is uniformly dispersed, prevent local DMSO
Concentration is excessive and the excessively multipair cell of exothermic dissolution causes damage.Experimental group is divided into two groups, and one group with the full culture containing 1.5g/LBDT
Base dispels cell, takes 0.5ml cell suspending liquids to be placed in 2ml cryopreservation tubes, and 5min is balanced under ice-water bath (4 DEG C), treats that cell hangs
Supernatant liquid is changed into being added dropwise the culture medium containing 16vol%DMSO of isometric (0.5ml) after gel state；Another group at ice-water bath (4 DEG C)
Lower balance 5min, is first added dropwise the full culture medium of 0.25ml trehaloses containing 0.2mol/L after cell suspending liquid is changed into gel state, drop
Add and be added dropwise full culture mediums of the 0.25ml containing 32vol%DMSO again after finishing, other operation all sames, three groups freeze system most
Final concentration of control group 8vol%DMSO, experimental group 0.75g/L BDT, 8vol%DMSO (BDT-DMSO groups) and 0.75g/L
BDT, 8vol%DMSO, 0.05mol/L trehalose (BDT-DMSO-Trehalose groups), the cell density frozen be 1 ×
106cells/mL.Cryopreservation tube is positioned over program temperature reduction box dripping cryoprotector and freezing system and reach after swelling equilibrium
In, cooling box is placed in -80 DEG C of refrigerator, and program temperature reduction box can make rate of temperature fall be maintained at 1 DEG C/min.In -80 DEG C of refrigerators
After middle placement 24h, freezing storing box is taken out, three groups of cryopreservation tubes are moved into liquid nitrogen and continue to freeze 3 days.
Embodiment 4
The rewarming process of schwann cell：
After being preserved 3 days in liquid nitrogen, cryopreservation tube is taken out, 5min is shaken in 37 DEG C of waters bath with thermostatic control, it is ensured that gel state changes
For liquid.Cell suspending liquid in cryopreservation tube is moved on in centrifuge tube, appropriate culture medium 1000r/m centrifugations 8min is added and removes supernatant,
In order to thoroughly remove the gelator and cryoprotector in cell suspending liquid, this step is repeated twice.After rewarming, cell is adjusted
Density is 6000cells/mL, is seeded cells into 96 orifice plates, is placed in 37 DEG C, 5%CO2Incubator in cultivate, after wait
Continuous experiment.Above-mentioned all operations are carried out in clean bench, it is ensured that strict gnotobasis.
Embodiment 5
The detection of schwann cell：
Cell survival rate：Detected using Apoptosis with downright bad detection kit.Fluorescence dye is carried out to the cell after rewarming
Color, and in fluorescence microscopy Microscopic observation, can intuitively find out the survival condition of cell.This experiment uses Hoechst 33342
With propidium iodide (PI) double colouring methods.Hoechst 33342 can penetrate the cell membrane of normal cell, normal cell and apoptosis
Cell can be colored, and the fluorescence reaction of apoptotic cell can be more obvious than normal cell after dyeing.Propidium iodide (PI) can not be penetrated
Cell membrane, therefore normal cell can not dye by PI, and for non-viable non-apoptotic cell, the integrality of its cell membrane is lost, propidium iodide
(PI) it can be dyed into cell.Therefore normal cell shows as blue-fluorescence, and non-viable non-apoptotic cell shows as blue-fluorescence+red
Color fluorescence, can significantly distinguish the life or death of cell.
Schwann cell density is adjusted to 6000cells/mL after rewarming, is inoculated into per the μ l of hole 100 in 96 orifice plates, every group is pressed
Freezing mode is different to be inoculated with six holes respectively, and 96 orifice plates then are placed in into 37 DEG C, 5%CO2Incubator in culture 3 days after, inhale
The supernatant in 96 orifice plates is walked, is washed twice with PBS, the dyeing liquor that 200 μ l are prepared then is added per hole, 4 DEG C are positioned over
It is incubated after 20-30min in fluorescence microscopy Microscopic observation red and blue-fluorescence and photographs to record, schwann cell survival rate in environment
As a result such as Fig. 5.
The activity of cell and increment：Tested using MTT colorimetric methods.MTT experiment is a kind of detection cell survival and growth
Method, MTT is a kind of developer, and concrete structure is 3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide smelling salts, and it shows
Chromogen reason can aoxidize the succinate dehydrogenase in living cells mitochondria for MTT, itself be changed into water insoluble bluish violet crystallization first
A ceremonial jade-ladle, used in libation, and be deposited in cell, and dead cell is without this function.First a ceremonial jade-ladle, used in libation can be dissolved by dimethyl sulfoxide (DMSO) (DMSO), with ELIASA in spy
Its light absorption value is determined under standing wave length, living cells quantity can be reflected indirectly.In cell quantity and suitable Detection wavelength, light absorption value
Size is directly proportional to cell number.
Schwann cell density is adjusted to 6000cells/mL after rewarming, is inoculated into per the μ l of hole 100 in 96 orifice plates, every group is pressed
Freezing mode is different to be inoculated with six holes respectively, connects 3 piece of 96 orifice plate, 96 orifice plates then are placed in into 37 DEG C, 5%CO2Incubator in
Culture.One piece of 96 orifice plate is taken out when culture 12h, 1 day, 2 days, 3 days, 5 days, the MTT that 20 μ l of addition have been configured per hole is molten
Liquid, is subsequently placed into 37 DEG C, 5%CO2Incubator in cultivate.96 orifice plates are taken out after 4h, supernatant is siphoned away, 150 μ l are added per hole
DMSO, shakes 5-10min, after purple crystal fully dissolves, 96 orifice plates is placed in ELIASA, first determines one with continuous wavelength
Individual hole absorption curve, finds out the corresponding wavelength of peak value of absorption, then determines absorbance with the wavelength.In incubation, if
Culture medium color yellowing needs to change the growing environment that liquid ensures cell in time in 96 orifice plates.
Test result continues to freeze cell after recovery in 3 days as shown in fig. 6, freezing and being transferred to after 24h in liquid nitrogen at -80 DEG C
In 12h, 1 day, 2 days, 3 days, the MTT testing results of 5 days.The form for the schwann cell that this method freezes and fresh schwann cell
Basic indifference, and survival rate is up to 98.59% and energy normal proliferative, it was demonstrated that frozen using the NEW TYPE OF COMPOSITE cryopreservation system
Schwann cell can keep its original biological characteristics.After method provided by the present invention freezing schwann cell,
The use of freezing protective agent is not only reduced during freezing, murder by poisoning of the toxic reagent to cell is reduced, and change cold
Freeze protectant addition manner, cryoprotector is added dropwise after gel is formed and reaches swelling equilibrium, cryoprotection is reduced
Osmotic injury caused by agent, while freezing the reduction of system freezing point, reduces the freezing injury that schwann cell is subject to；In rewarming
During, because the supermolecular gel that amino acids gelator is self-assembly of has thermal reversibility, gel conversion is liquid,
It is easy to remove cryoprotector and amino acids gelator, it is ensured that the integrality of schwann cell.
Claims (5)
1. compound cryosar freezes the method that system freezes schwann cell, it is characterised in that comprise the following steps：
1) by amino acids gelator heating be dissolved in cell culture medium, filtering, it is degerming, with containing gelator cell train
Foster base prepares cell suspending liquid；
2) supermolecular gel is formed at 0-4 DEG C；
3) it is added dropwise and contains the protectant cell culture medium of composite frozen, the system for the treatment of reaches swelling equilibrium；The composite frozen protection
Agent is the mixing by permeability cryoprotector and impermeability cryoprotector；
4) it is put into cryopreservation tube to freeze in program freezing storing box, then will be frozen in cryopreservation tube immigration liquid nitrogen.
2. compound cryosar as claimed in claim 1 freezes the method that system freezes schwann cell, it is characterised in that the amino acid
The gellike factor is prepared as follows：
Dodecyl-Boc-L- methyl-P-tyrosines are prepared by alkylated reaction with Boc-L- methyl-P-tyrosines；
Dodecyl-Boc-L- methyl-P-tyrosines prepare dodecyl-Boc-L- tyrosine, as institute by hydrolysis
The amino acids gelator stated.
3. compound cryosar as claimed in claim 1 freezes the method that system freezes schwann cell, it is characterised in that described cell
Culture medium is cultivated entirely for the cell being prepared into after dual anti-of addition 10vol% hyclones and 1vol% in RPMI1640 culture mediums
Base.
4. compound cryosar as claimed in claim 1 freezes the method that system freezes schwann cell, it is characterised in that described infiltration
Property cryoprotector be dimethyl sulfoxide (DMSO), impermeability cryoprotector is trehalose, the two final concentration is respectively 7.5~
8.5vol% and 0.045~0.055mol/L.
5. compound cryosar as claimed in claim 1 freezes the method that system freezes schwann cell, it is characterised in that step 3 is first added dropwise
Cell culture medium containing impermeability cryoprotector, then the cell culture medium containing permeability cryoprotector is added dropwise.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110004107A (en) * | 2019-05-17 | 2019-07-12 | 西北工业大学 | The method that a kind of pretreatment of cell surface and cell directional thaw |
| CN112167243A (en) * | 2020-10-14 | 2021-01-05 | 中国科学技术大学 | Erythrocyte cryopreservation liquid and rapid cryopreservation method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104145944A (en) * | 2014-08-25 | 2014-11-19 | 山东省海洋生物研究院 | Ultralow-temperature cryopreservation and activation method of sperm of scapharca broughtonii sckrenck |
| CN105454221A (en) * | 2016-01-13 | 2016-04-06 | 武汉理工大学 | Method for utilizing micro-channel for cryopreserving rat islet cells at low temperature |
-
2017
- 2017-04-21 CN CN201710266580.2A patent/CN106982820B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104145944A (en) * | 2014-08-25 | 2014-11-19 | 山东省海洋生物研究院 | Ultralow-temperature cryopreservation and activation method of sperm of scapharca broughtonii sckrenck |
| CN105454221A (en) * | 2016-01-13 | 2016-04-06 | 武汉理工大学 | Method for utilizing micro-channel for cryopreserving rat islet cells at low temperature |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110004107A (en) * | 2019-05-17 | 2019-07-12 | 西北工业大学 | The method that a kind of pretreatment of cell surface and cell directional thaw |
| CN110004107B (en) * | 2019-05-17 | 2023-06-02 | 西北工业大学 | Cell surface pretreatment and cell directional thawing method |
| CN112167243A (en) * | 2020-10-14 | 2021-01-05 | 中国科学技术大学 | Erythrocyte cryopreservation liquid and rapid cryopreservation method |
| CN112167243B (en) * | 2020-10-14 | 2023-03-10 | 中国科学技术大学 | Erythrocyte cryopreservation liquid and rapid cryopreservation method |
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