CN103891709A - Cell cryopreservation liquid and cell cryopreservation method - Google Patents
Cell cryopreservation liquid and cell cryopreservation method Download PDFInfo
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Abstract
The invention relates to a cell cryopreservation liquid and a cell cryopreservation method. The cell cryopreservation liquid comprises 90-95% of a whole cell culture liquid and 5-10% of dimethyl sulfoxide according to the volume percentage, wherein the whole cell culture liquid contains a cell culture medium and fetal calf serum with the volume ratio of 9:1. The whole cell culture liquid of the cell cryopreservation liquid provides essential nutrient substances for cellular life metabolism, and the dimethyl sulfoxide is used as an antifreezing agent; through reasonable proportion of the amounts of the cell culture medium and the fetal calf serum in the whole cell culture liquid and the amounts of the whole cell culture liquid and the antifreezing agent, the whole cell culture liquid with lower content of the fetal calf serum can provide the essential and enough nutrient substances for the cellular life metabolism; moreover, the appropriate amount of the antifreezing agent can prevent or reduce damage effects of frozen ice crystals on cells so as to improve the survival rate of the cells; and the cell cryopreservation liquid contains no serum easily causing pollution, thereby having lower pollution risk.
Description
Technical field
The present invention relates to cell cryopreservation technology, particularly relate to a kind of cells frozen storing liquid and cell freezing method.
Background technology
Cell cryopreservation and recovery are one of important technologies in cell culture technology, and cell cryopreservation is a kind of method of preserving cultured cell.The basic principle of cell cryopreservation and recovery is to freeze soon and melt slowly, can preserve to greatest extent like this cell viability.
A kind of solution that must use when cells frozen storing liquid is cell cryopreservation, its effect is that cell frozen needs is suspended in to cryopreserving liquid, supplies with the necessary nutriment of cell life metabolism, can prevent or reduce the damaging action of freezing ice crystal to cell simultaneously.For enough nutrition is provided for cell, keep the survival rate of cell, existing cells frozen storing liquid generally adds the nutriment of more serum as Growth of Cells, and the volume that serum accounts for cells frozen storing liquid is not generally from 20% ~ 90% etc., not only expensive, also easily cause pollution risk higher.
Summary of the invention
Based on this, be necessary to provide a kind of and improve cell survival rate, and the lower cells frozen storing liquid of pollution risk.
Further, provide a kind of cell freezing method.
A kind of cells frozen storing liquid, by volume percentage comprises following component:
Full cell culture fluid 90 ~ 95%;
Dimethyl sulfoxide (DMSO) 5 ~ 10%;
Wherein, described full cell culture fluid comprises that volume ratio is cell culture medium and the hyclone of 9:1.
In an embodiment, by volume percentage comprises following component therein:
Full cell culture fluid 90%;
Dimethyl sulfoxide (DMSO) 10%;
Wherein, described full cell culture fluid comprises that volume ratio is cell culture medium and the hyclone of 9:1.
In an embodiment, described cell culture medium comprises following component therein:
Anhydrous calcium chloride 265.00mg/L; Ferric nitrate 0.10mg/L; Potassium chloride 400.00mg/L; Anhydrous magnesium sulfate 97.67mg/L; Sodium chloride 6400.00mg/L; AMSP 109.00mg/L; Succinic acid 75.00mg/L; Sodium succinate 100.00mg/L; L-R-gene 84.00mg/L; L-hydrochloric acid cystine 63.00mg/L; Glycine 30.00mg/L; L-histidine monohydrochloride 42.00mg/L; ILE 105.00mg/L; L-Leu 105.00mg/L; LYS 146.00mg/L; METHIONINE 30.00mg/L; L-Phe 66.00mg/L; Serine 42.00mg/L; L-threonine 95.00mg/L; L-Trp 16.00mg/L; TYR 72.00mg/L; Valine 94.00mg/L; D-VB5 calcium 4.00mg/L; Choline tartrate 7.20mg/L; Folic acid mg/L; Inositol 7.20mg/L; Vitamin PP 4.00mg/L; Vitamin b3 0.40mg/L; Thiamine hydrochloride 4.00mg/L; Pyridoxine hydrochloride 4.00mg/L; Glucose 1000.00mg/L; Sodium Pyruvate 110.00mg/L and phenol red 9.30mg/L.
In an embodiment, described cell culture medium comprises following component therein:
Calcium chloride 116.6mg/L; Copper sulphate 0.0013mg/L; Fe(NO3)39H2O 0.05mg/L; Green vitriol 0.417mg/L; Potassium chloride 311.8mg/L; Magnesium chloride 28.64mg/L; Anhydrous magnesium sulfate 48.84mg/L; Sodium chloride 6999.6mg/L; AMSP 54.35mg/L; ADSP 71.02mg/L; Zinc vitriol 0.432mg/L; L-R-gene 147.5mg/L; CYSTINE hydrochloride 31.29mg/L; Glu 365mg/L; Glycine 18.75mg/L; L-histidine monohydrochloride 31.48mg/L; ILE 54.47mg/L; L-Leu 59.05mg/L; LYS 91.25mg/L; METHIONINE 17.24mg/L; L-Phe 35.48mg/L; Serine 26.25mg/L; L-threonine 53.45mg/L; ALANINE 4.45mg/L; L-asparagine 7.5mg/L; L-ASPARTIC ACID 6.65mg/L; L-cysteine hydrochloride 17.56mg/L; Pidolidone 7.35mg/L; L-PROLINE 17.25mg/L; L-Trp 9.02mg/L; TYR mg/L; Valine 52.85mg/L; DEXTROSE ANHYDROUS 3151mg/L; Hypoxanthine 2mg/L; Linoleic acid 0.042mg/L; Lipoic acid 0.105mg/L; Phenol red 8.1mg/L; Putriscine dihydrochloride 0.081mg/L; Sodium Pyruvate 55mg/L; Biotin 0.0035mg/L; D-VB5 calcium 2.24mg/L; Choline Chloride 8.98mg/L; Folic acid 2.65mg/L; Inositol 12.6mgL; Vitamin PP 2.02mg/L; Pyridoxal hydrochloride 2mg/L; Puridoxine hydrochloride 0.03mgL; Vitamin b3 0.219mg/L; Thiamine hydrochloride 2.17mg/L; Thymidine 0.365mg/L and Cobastab
120.68mg/L.
In an embodiment, described cell culture medium comprises following component therein:
Four water-calcium nitrate 100mg/L; Potassium chloride 400mg/L; Anhydrous magnesium sulfate 48.84mg/L; Sodium chloride 6000mg/L; ADSP 800mg/L; L-R-gene 200mg/L; L-asparagine 50mg/L; L-ASPARTIC ACID 20mg/L; CYSTINE hydrochloride 65.15mg/L; Pidolidone 20mg/L; Glu 300mg/L; Glycine 10mg/L; L-histidine monohydrochloride 15mg/L; L-hydroxyproline 20mg/L; ILE 50mg/L; LYS 40mg/L; METHIONINE 15mg/L; L-Phe 15mg/L; L-PROLINE 20mg/L; Serine 30mg/L; L-threonine 20mg/L; L-Trp 5mg/L; TYR 20mg/L; Valine 20mg/L; DEXTROSE ANHYDROUS 2000mg/mL; Reduced glutathione 1mg/L; Phenol red 5mg/L; Bio 0.2mg/L; Calcium pantothenate 0.25mg/L; Choline Chloride 3mg/L; Folic acid 1mg/L; Inositol 35mg/L; Vitamin PP 1mg/L; P-aminobenzoic acid 1mg/L; Cobastab
61mg/L; Cobastab
20.2mg/L; Cobastab
11mg/L and Cobastab
120.005mg/L.
A kind of cell freezing method, comprises the steps:
The cell of logarithmic phase is washed with PBS buffer solution, and the cell of the logarithmic phase after described washing is suspended with above-mentioned cells frozen storing liquid, then carry out cell counting, and dilute with described cells frozen storing liquid, obtaining concentration is 0.5 ~ 5 × 10
6the cell of logarithmic phase and the mixed liquor of described cells frozen storing liquid of individual/milliliter;
Be 0.5 ~ 5 × 10 by described concentration
6the cell of logarithmic phase and the mixed liquor of described cells frozen storing liquid of individual/milliliter are sub-packed in the aseptic cryopreservation tube of 1mL;
Described in inciting somebody to action, concentration being housed is 0.5 ~ 5 × 10
6individual/the cell of logarithmic phase of milliliter and the aseptic cryopreservation tube of the mixed liquor of described cells frozen storing liquid are positioned in program temperature reduction box and are cooled to-80 DEG C, and at-80 DEG C, place 24 hours ~ 96 hours, then described aseptic cryopreservation tube are transferred in liquid nitrogen and are preserved.
Therein in an embodiment, described cell is attached cell, the described step that the cell of logarithmic phase is washed with PBS buffer solution is: described attached cell is cultured to after logarithmic phase, remove the cell culture fluid in culture dish, add PBS buffer solution to wash, then remove described PBS buffer solution.
Therein in an embodiment, after the step that the cell of logarithmic phase is washed with PBS buffer solution, also comprise the step of cell dissociation, the step of described cell dissociation is: adding 0.5 ~ 2mL concentration to the surface of described attached cell is 0.25% trypsin solution, in 37 DEG C of incubators, digest 2 ~ 5 minutes, add again the full cell culture fluid of cells frozen storing liquid described in 3 ~ 5mL, to stop described tryptic digestion, then described attached cell is dispelled, and by the mixed liquor of described attached cell and described full cell culture fluid under 500 revs/min ~ 1000 revs/min centrifugal 3 minutes ~ 10 minutes, supernatant discarded obtains cell precipitation.
Therein in an embodiment, in described trypsin solution, contain concentration and be 0.38% ethylenediamine tetra-acetic acid.
Therein in an embodiment, described cell is suspension cell, described the cell of logarithmic phase is specially by the step that PBS buffer solution washs: by described suspension cell culture to logarithmic phase, by the cell culture fluid of the cell that contains logarithmic phase under 500 revs/min ~ 1000 revs/min centrifugal 3 minutes ~ 10 minutes, supernatant discarded obtains cell precipitation, add PBS buffer solution, under 500 revs/min ~ 1000 revs/min centrifugal 3 minutes ~ 10 minutes again, supernatant discarded obtained the cell precipitation after washing.
Full cell culture fluid in above-mentioned cells frozen storing liquid provides necessary nutriment for the metabolism of cell life, dimethyl sulfoxide (DMSO) is as antifreezing agent, by the amount of cell culture medium and hyclone in the full cell culture fluid of rational proportion and the amount of full cell culture fluid and antifreezing agent, make the full cell culture fluid that hyclone content is lower to provide necessary for the metabolism of cell life, enough nutriments, and the amount of suitable antifreezing agent can prevent or reduce the damaging action of freezing ice crystal to cell, improve the survival rate of cell, and this cells frozen storing liquid is not containing the serum easily polluting, pollution risk is lower.
Brief description of the drawings
Fig. 1 is the cell freezing method flow chart of an embodiment.
Embodiment
For above-mentioned purpose of the present invention, feature and advantage can be become apparent more, below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in detail.A lot of details are set forth in the following description so that fully understand the present invention.But the present invention can implement to be much different from alternate manner described here, and those skilled in the art can do similar improvement without prejudice to intension of the present invention in the situation that, and therefore the present invention is not subject to the restriction of following public concrete enforcement.
The cells frozen storing liquid of one embodiment, by volume percentage comprises following component: full cell culture fluid 90 ~ 95% and dimethyl sulfoxide (DMSO) 5 ~ 10%.
Full cell culture fluid comprises cell culture medium and hyclone, and the volume ratio of cell culture medium and hyclone is 9:1.
Full cell culture fluid provides necessary nutriment for the metabolism of cell life.Dimethyl sulfoxide (DMSO), as antifreezing agent, can improve the permeability of cell membrane to water, is beneficial to outside intracellular moisture emigrated cell, reduces the formation of intracellular ice crystal, thereby reduces the cellular damage causing due to ice crystal.
Above-mentioned cells frozen storing liquid is by the amount of the cell culture fluid in the full cell culture fluid of rational proportion nutriment and hyclone and the amount of full cell culture fluid and antifreezing agent dimethyl sulfoxide (DMSO), can also the cellular damage causing due to ice crystal can be reduced for cell provides necessary nutriment, thereby the survival rate of cell can be improved.And because this nutriment does not contain expensive serum, pollution risk is lower, cost is lower simultaneously, is conducive to reduce frozen cost.
Preferably, the percentage by volume of full cell culture fluid is 90%, and the percentage by volume of dimethyl sulfoxide (DMSO) is 10%.
Preferably, cell culture medium comprises following component:
Calcium chloride 116.6mg/L; Copper sulphate 0.0013mg/L; Fe(NO3)39H2O 0.05mg/L; Green vitriol 0.417mg/L; Potassium chloride 311.8mg/L; Magnesium chloride 28.64mg/L; Anhydrous magnesium sulfate 48.84mg/L; Sodium chloride 6999.6mg/L; AMSP 54.35mg/L; ADSP 71.02mg/L; Zinc vitriol 0.432mg/L; L-R-gene 147.5mg/L; CYSTINE hydrochloride 31.29mg/L; Glu 365mg/L; Glycine 18.75mg/L; L-histidine monohydrochloride 31.48mg/L; ILE 54.47mg/L; L-Leu 59.05mg/L; LYS 91.25mg/L; METHIONINE 17.24mg/L; L-Phe 35.48mg/L; Serine 26.25mg/L; L-threonine 53.45mg/L; ALANINE 4.45mg/L; L-asparagine 7.5mg/L; L-ASPARTIC ACID 6.65mg/L; L-cysteine hydrochloride 17.56mg/L; Pidolidone 7.35mg/L; L-PROLINE 17.25mg/L; L-Trp 9.02mg/L; TYR mg/L; Valine 52.85mg/L; DEXTROSE ANHYDROUS 3151mg/L; Hypoxanthine 2mg/L; Linoleic acid 0.042mg/L; Lipoic acid 0.105mg/L; Phenol red 8.1mg/L; Putriscine dihydrochloride 0.081mg/L; Sodium Pyruvate 55mg/L; Biotin 0.0035mg/L; D-VB5 calcium 2.24mg/L; Choline Chloride 8.98mg/L; Folic acid 2.65mg/L; Inositol 12.6mg/L; Vitamin PP 2.02mg/L; Pyridoxal hydrochloride 2mg/L; Puridoxine hydrochloride 0.03mgL; Vitamin b3 0.219mg/L; Thiamine hydrochloride 2.17mg/L; Thymidine 0.365mg/L and Cobastab
120.68mg/L.
In other embodiments, cell culture medium comprises following component: calcium chloride 116.6mg/L; Copper sulphate 0.0013mg/L; Fe(NO3)39H2O 0.05mg/L; Green vitriol 0.417mg/L; Potassium chloride 311.8mg/L; Magnesium chloride 28.64mg/L; Anhydrous magnesium sulfate 48.84mg/L; Sodium chloride 6999.6mg/L; AMSP 54.35mg/L; ADSP 71.02mg/L; Zinc vitriol 0.432mg/L; L-R-gene 147.5mg/L; CYSTINE hydrochloride 31.29mg/L; Glu 365mg/L; Glycine 18.75mg/L; L-histidine monohydrochloride 31.48mg/L; ILE 54.47mg/L; L-Leu 59.05mg/L; LYS 91.25mg/L; METHIONINE 17.24mg/L; L-Phe 35.48mg/L; Serine 26.25mg/L; L-threonine 53.45mg/L; ALANINE 4.45mg/L; L-asparagine 7.5mg/L; L-ASPARTIC ACID 6.65mg/L; L-cysteine hydrochloride 17.56mg/L; Pidolidone 7.35mg/L; L-PROLINE 17.25mg/L; L-Trp 9.02mg/L; TYR mg/L; Valine 52.85mg/L; DEXTROSE ANHYDROUS 3151mg/L; Hypoxanthine 2mg/L; Linoleic acid 0.042mg/L; Lipoic acid 0.105mg/L; Phenol red 8.1mg/L; Putriscine dihydrochloride 0.081mg/L; Sodium Pyruvate 55mg/L; Biotin 0.0035mg/L; D-VB5 calcium 2.24mg/L; Choline Chloride 8.98mg/L; Folic acid 2.65mg/L; Inositol 12.6mg/L; Vitamin PP 2.02mg/L; Pyridoxal hydrochloride 2mg/L; Puridoxine hydrochloride 0.03mg/L; Vitamin b3 0.219mg/L; Thiamine hydrochloride 2.17mg/L; Thymidine 0.365mg/L and Cobastab
120.68mg/L.
In other embodiment, cell culture medium comprises following component:
Four water-calcium nitrate 100mg/L; Potassium chloride 400mg/L; Anhydrous magnesium sulfate 48.84mg/L; Sodium chloride 6000mg/L; ADSP 800mg/L; L-R-gene 200mg/L; L-asparagine 50mg/L; L-ASPARTIC ACID 20mg/L; CYSTINE hydrochloride 65.15mg/L; Pidolidone 20mg/L; Glu 300mg/L; Glycine 10mg/L; L-histidine monohydrochloride 15mg/L; L-hydroxyproline 20mg/L; ILE 50mg/L; LYS 40mg/L; METHIONINE 15mg/L; L-Phe 15mg/L; L-PROLINE 20mg/L; Serine 30mg/L; L-threonine 20mg/L; L-Trp 5mg/L; TYR 20mg/L; Valine 20mg/L; DEXTROSE ANHYDROUS 2000mg/mL; Reduced glutathione 1mg/L; Phenol red 5mg/L; Bio 0.2mg/L; Calcium pantothenate 0.25mg/L; Choline Chloride 3mg/L; Folic acid 1mg/L; Inositol 35mg/L; Vitamin PP 1mg/L; P-aminobenzoic acid 1mg/L; Cobastab
61mg/L; Cobastab
20.2mg/L; Cobastab
11mg/L and Cobastab
120.005mg/L.
When cell culture medium in the full cell culture fluid of above-mentioned cells frozen storing liquid adopts respectively above-mentioned three kinds of different formulas, above-mentioned cells frozen storing liquid can be applicable to the various kinds of cell such as 293 cells, Hela cell, MCF-7 cell, Chinese hamster ovary celI, Raji cell, Daudi cell, HL60 cell, K562 cell, and applicability is extensive.
Refer to Fig. 1, a kind of cell freezing method, comprises the steps:
Step S110: the cell of logarithmic phase is washed with PBS buffer solution, and the cell of logarithmic phase after washing is suspended with above-mentioned cells frozen storing liquid, then carry out cell counting, and dilute with above-mentioned cells frozen storing liquid, obtaining concentration is 0.5 ~ 5 × 10
6the cell of logarithmic phase and the mixed liquor of cells frozen storing liquid of individual/milliliter.
The frozen cell of wish should be well-grown state, the cell cryopreservation of the phase of therefore taking the logarithm.Before cell cryopreservation, first use PBS buffer solution (phosphate buffer) to clean cell, to remove cell culture medium.
In the time that the frozen cell of wish is suspension cell, by suspension cell culture to logarithmic phase, by the cell culture fluid of the cell that contains logarithmic phase under 500 revs/min ~ 1000 revs/min centrifugal 3 minutes ~ 10 minutes, supernatant discarded obtains cell precipitation, then add PBS buffer solution re-suspended cell, under 500 revs/min ~ 1000 revs/min centrifugal 3 minutes ~ 10 minutes again, supernatant discarded obtained the cell precipitation after washing.Cell precipitation after washing is suspended with above-mentioned cells frozen storing liquid, then carry out cell counting, and dilute with above-mentioned cells frozen storing liquid, obtaining concentration is 0.5 ~ 5 × 10
6the cell of logarithmic phase and the mixed liquor of cells frozen storing liquid of individual/milliliter.
In the time that the frozen cell of wish is attached cell, this attached cell is cultured to after logarithmic phase, sops up the cell culture fluid in culture dish with liquid-transfering gun or pipette, in culture dish, add PBS buffer solution, rock and wash gently, then remove PBS buffer solution with liquid-transfering gun or pipette.
After washing, attached cell is still attached on culture dish, need to carry out cell dissociation, so that the attached cell on culture dish disperses.The step of cell dissociation is specially: adding 0.5 ~ 2mL concentration to the surface of attached cell is 0.25% trypsin solution, in 37 DEG C of incubators, digest 2 ~ 5 minutes, add again the full cell culture fluid of the above-mentioned cells frozen storing liquid of 3 ~ 5mL, to stop tryptic digestion, then attached cell is dispelled, and by the mixed liquor of attached cell and full cell culture fluid under 500 revs/min ~ 1000 revs/min centrifugal 3 minutes ~ 10 minutes, supernatant discarded obtained cell precipitation.This cell precipitation is suspended with above-mentioned cells frozen storing liquid, then carry out cell counting, and dilute with above-mentioned cells frozen storing liquid, obtaining concentration is 0.5 ~ 5 × 10
6the cell of logarithmic phase and the mixed liquor of cells frozen storing liquid of individual/milliliter.
Concentration be 0.25% trypsin solution for a certain amount of trypsase is dissolved in PBS buffer solution, wherein, the ratio of the volume of tryptic quality and PBS buffer solution is 0.25g:100mL.
Digestion is excessively larger to the activity damage of cell, and has part cell floating, runs off with the trypsase discarding; Digestion deficiency cell is difficult to blow down from culture dish, and piping and druming equally also can damaging cells activity repeatedly.Therefore the time of digestion is preferably 2 ~ 5 minutes.Digestion time is to become circle to observation of cell under microscope.
Preferably, adherent when too tight on culture dish when attached cell, be 0.38% ethylenediamine tetra-acetic acid (EDTA) for the ease of attached cell being disperseed, containing concentration in trypsin solution.The ratio of the volume of the PBS buffer solution in the quality of ethylenediamine tetra-acetic acid and trypsin solution is 0.038g:100mL.
Cell concentration is overstocked or cross rare survival rate that all can reduce cell, dilutes with above-mentioned cells frozen storing liquid, makes the concentration 0.5 ~ 5 × 10 of cell
6individual/milliliter, to ensure the survival rate of cell.
Step S120: be 0.5 ~ 5 × 10 by concentration
6the cell of logarithmic phase and the mixed liquor of cells frozen storing liquid of individual/milliliter are sub-packed in the aseptic cryopreservation tube of 1mL.
Be 0.5 ~ 5 × 10 by concentration
6the cell of logarithmic phase of individual/milliliter and the mixed liquor of cells frozen storing liquid be distributed in the aseptic cryopreservation tube of multiple 1mL, carry out respectively frozen, in the time that needs are recovered use, take out as required one or more frozen aseptic cryopreservation tubes of cell, can not cause the pollution of the aseptic cryopreservation tube to other frozen cells.
Step S130: it is 0.5 ~ 5 × 10 that concentration will be housed
6individual/the cell of logarithmic phase of milliliter and the multiple aseptic cryopreservation tube of the mixed liquor of above-mentioned cells frozen storing liquid are positioned in program temperature reduction box and are cooled to-80 DEG C, and at-80 DEG C, place 24 hours ~ 96 hours, then multiple aseptic cryopreservation tubes are transferred in liquid nitrogen and are preserved.
It is 0.5 ~ 5 × 10 that concentration is housed
6individual/the cell of logarithmic phase of milliliter and the aseptic cryopreservation tube of the mixed liquor of above-mentioned cells frozen storing liquid are positioned at the uniform velocity cooling in program temperature reduction box, are conducive to improve the survival rate of cell.
It is 0.5 ~ 5 × 10 that concentration is housed
6individual/the cell of logarithmic phase of milliliter and the aseptic cryopreservation tube of the mixed liquor of above-mentioned cells frozen storing liquid are cooled to after-80 DEG C in program temperature reduction box, place 24 hours ~ 96 hours at-80 DEG C, are then transferred in liquid nitrogen and preserve.
In the time carrying out cell recovery, first from liquid nitrogen, take out the aseptic cryopreservation tube of required quantity, aseptic cryopreservation tube is put into 37 DEG C of water-baths immediately, shake was melted freeze-stored cell in 1 ~ 2 minute; Then under 500 ~ 1000 revs/min centrifugal 3 ~ 10 minutes, abandon supernatant; Add the above-mentioned full cell culture fluid re-suspended cell of 1ml, this cell and the suspension of full cell culture fluid are added in the above-mentioned full cell culture fluid of 8 ~ 12ml, at 37 DEG C, leave standstill and cultivate 16 ~ 24 hours.
Cultivate after 16 ~ 24 hours, the state under microscope after observation of cell recovery, changes full cell culture fluid, continues to cultivate the cultivation of going down to posterity after 2 ~ 3 days.
In the time that cell is attached cell, in micro-Microscopic observation, after more than 80% cell attachment growth, change again full cell culture fluid.
Above-mentioned cell freezing method uses and comprises that volume fraction is that the cells frozen storing liquid of 90 ~ 95% full cell culture fluids and the volume fraction DMSO that is 5 ~ 10% carries out freeze-stored cell, is conducive to improve the survival rate of cell, and reduces pollution risk.
And full cell culture fluid comprises that volume ratio is cell culture medium and the hyclone of 9:1, and the volume fraction of hyclone in cells frozen storing liquid is only 9 ~ 9.5%, and content is lower, uses the lower cells frozen storing liquid of serum content, and frozen cost is low.
It is below specific embodiment.
Embodiment 1
1,293 cells are cultured to after logarithmic phase, under gnotobasis, sop up in the cell culture fluid in culture dish with electric pipette, in culture dish, add 10mL PBS buffer solution, rock and wash gently, then remove PBS buffer solution with electric pipette;
2, under gnotobasis, adding 0.5mL concentration to the surface uniform of 293 cells in culture dish is 0.25% trypsin solution, in 37 DEG C of incubators, digest 2 minutes, in culture dish, add the full cell culture fluid of 3mL, stop tryptic digestion, then with electric pipette, 293 cells are dispelled and get off to make 293 cells to be suspended in above-mentioned full cell culture fluid from culture dish; Wherein, above-mentioned full cell culture fluid comprises that volume ratio is cell culture medium and the hyclone of 9:1, and cell culture medium comprises following component:
Anhydrous calcium chloride 265.00mg/L; Ferric nitrate 0.10mg/L; Potassium chloride 400.00mg/L; Anhydrous magnesium sulfate 97.67mg/L; Sodium chloride 6400.00mg/L; AMSP 109.00mg/L; Succinic acid 75.00mg/L; Sodium succinate 100.00mg/L; L-R-gene 84.00mg/L; L-hydrochloric acid cystine 63.00mg/L; Glycine 30.00mg/L; L-histidine monohydrochloride 42.00mg/L; ILE 105.00mg/L; L-Leu 105.00mg/L; LYS 146.00mg/L; METHIONINE 30.00mg/L; L-Phe 66.00mg/L; Serine 42.00mg/L; L-threonine 95.00mg/L; L-Trp 16.00mg/L; TYR 72.00mg/L; Valine 94.00mg/L; D-VB5 calcium 4.00mg/L; Choline tartrate 7.20mg/L; Folic acid mg/L; Inositol 7.20mg/L; Vitamin PP 4.00mg/L; Vitamin b3 0.40mg/L; Thiamine hydrochloride 4.00mg/L; Pyridoxine hydrochloride 4.00mg/L; Glucose 1000.00mg/L; Sodium Pyruvate 110.00mg/L and phenol red 9.30mg/L;
3,293 cells and the suspension of full cell culture fluid are transferred in the aseptic centrifuge tube of 15ml, under 500 revs/min centrifugal 10 minutes, abandoning supernatant obtains 293 cell precipitations, this 293 cell precipitation is suspended with cells frozen storing liquid, then carry out cell counting, and dilute with above-mentioned cells frozen storing liquid, obtaining concentration is 0.5 × 10
6293 cells of the logarithmic phase of individual/milliliter and the mixed liquor of cells frozen storing liquid; Wherein, above-mentioned cells frozen storing liquid by volume percentage comprise 90% above-mentioned full cell culture fluid and 10% dimethyl sulfoxide (DMSO);
4, be 0.5 × 10 by concentration
6293 cells of logarithmic phase and the mixed liquor of cells frozen storing liquid of individual/milliliter are sub-packed in the aseptic cryopreservation tube of 1mL;
5, concentration will be housed is 0.5 × 10
6individual/293 cells of logarithmic phase of milliliter and the multiple aseptic cryopreservation tube of the mixed liquor of above-mentioned cells frozen storing liquid are positioned over program temperature reduction box (Nalgene, article No.: 5100-0001C) in, at-80 DEG C, place 24 hours, then multiple aseptic cryopreservation tubes are transferred in liquid nitrogen and are preserved.
After frozen one month, carry out cell recovery, first from liquid nitrogen, take out 1 aseptic cryopreservation tube, this aseptic cryopreservation tube is put into 37 DEG C of water-baths immediately, shake was melted freeze-stored cell in 1 minute; Then under 500 revs/min centrifugal 10 minutes, abandon supernatant; Add the above-mentioned full cell culture fluid re-suspended cell of 1ml, this cell and the suspension of full cell culture fluid are added in the above-mentioned full cell culture fluid of 8ml, at 37 DEG C, leave standstill and cultivate 24 hours, measure the survival rate of cell, survival rate is 90%.
Embodiment 2
1, MCF-7 cell is cultured to after logarithmic phase, under gnotobasis, sops up in the cell culture fluid in culture dish with electric pipette, in culture dish, add 15mL PBS buffer solution, rock and wash gently, then remove PBS buffer solution with electric pipette;
2, under gnotobasis, adding 2mL concentration to the surface uniform of the MCF-7 cell in culture dish is 0.25% trypsin solution, in 37 DEG C of incubators, digest 5 minutes, in culture dish, add the full cell culture fluid of 5mL, stop tryptic digestion, then with electric pipette, MCF-7 cell is dispelled and gets off to make MCF-7 cell to be suspended in above-mentioned full cell culture fluid from culture dish; Wherein, above-mentioned full cell culture fluid comprises that volume ratio is cell culture medium and the hyclone of 9:1, and cell culture medium comprises following component:
Calcium chloride 116.6mg/L; Copper sulphate 0.0013mg/L; Fe(NO3)39H2O 0.05mg/L; Green vitriol 0.417mg/L; Potassium chloride 311.8mg/L; Magnesium chloride 28.64mg/L; Anhydrous magnesium sulfate 48.84mg/L; Sodium chloride 6999.6mg/L; AMSP 54.35mg/L; ADSP 71.02mg/L; Zinc vitriol 0.432mg/L; L-R-gene 147.5mg/L; CYSTINE hydrochloride 31.29mg/L; Glu 365mg/L; Glycine 18.75mg/L; L-histidine monohydrochloride 31.48mg/L; ILE 54.47mg/L; L-Leu 59.05mg/L; LYS 91.25mg/L; METHIONINE 17.24mg/L; L-Phe 35.48mg/L; Serine 26.25mg/L; L-threonine 53.45mg/L; ALANINE 4.45mg/L; L-asparagine 7.5mg/L; L-ASPARTIC ACID 6.65mg/L; L-cysteine hydrochloride 17.56mg/L; Pidolidone 7.35mg/L; L-PROLINE 17.25mg/L; L-Trp 9.02mg/L; TYR mg/L; Valine 52.85mg/L; DEXTROSE ANHYDROUS 3151mg/L; Hypoxanthine 2mg/L; Linoleic acid 0.042mg/L; Lipoic acid 0.105mg/L; Phenol red 8.1mg/L; Putriscine dihydrochloride 0.081mg/L; Sodium Pyruvate 55mg/L; Biotin 0.0035mg/L; D-VB5 calcium 2.24mg/L; Choline Chloride 8.98mg/L; Folic acid 2.65mg/L; Inositol 12.6mg/L; Vitamin PP 2.02mg/L; Pyridoxal hydrochloride 2mg/L; Puridoxine hydrochloride 0.03mgL; Vitamin b3 0.219mg/L; Thiamine hydrochloride 2.17mg/L; Thymidine 0.365mg/L and Cobastab
120.68mg/L;
3, MCF-7 cell and the suspension of full cell culture fluid are transferred in the aseptic centrifuge tube of 15ml, under 1000 revs/min centrifugal 3 minutes, abandoning supernatant obtains MCF-7 cell precipitation, this MCF-7 cell precipitation is suspended with cells frozen storing liquid, then carry out cell counting, and dilute with above-mentioned cells frozen storing liquid, obtaining concentration is 5 × 10
6the MCF-7 cell of the logarithmic phase of individual/milliliter and the mixed liquor of cells frozen storing liquid; Wherein, above-mentioned cells frozen storing liquid by volume percentage comprise 95% above-mentioned full cell culture fluid and 5% dimethyl sulfoxide (DMSO);
4, be 5 × 10 by concentration
6the MCF-7 cell of logarithmic phase and the mixed liquor of cells frozen storing liquid of individual/milliliter are sub-packed in the aseptic cryopreservation tube of 1mL;
5, concentration will be housed is 5 × 10
6individual/MCF-7 the cell of logarithmic phase of milliliter and the multiple aseptic cryopreservation tube of the mixed liquor of above-mentioned cells frozen storing liquid are positioned over program temperature reduction box (Nalgene, article No.: 5100-0001C) in, at-80 DEG C, place 96 hours, then multiple aseptic cryopreservation tubes are transferred in liquid nitrogen and are preserved.
After frozen 3 months, carry out cell recovery, first from liquid nitrogen, take out 1 aseptic cryopreservation tube, this aseptic cryopreservation tube is put into 37 DEG C of water-baths immediately, shake was melted freeze-stored cell in 1.5 minutes; Then under 1000 revs/min centrifugal 3 minutes, abandon supernatant; Add the above-mentioned full cell culture fluid re-suspended cell of 1ml, this cell and the suspension of full cell culture fluid are added in the above-mentioned full cell culture fluid of 12ml, at 37 DEG C, leave standstill and cultivate 16 hours, measure the survival rate of cell, survival rate is 85%.
Embodiment 3
1, HL60 cell is cultured to after logarithmic phase, by the cell culture fluid of the cell that contains logarithmic phase centrifugal 6min under 800 revs/min, supernatant discarded obtains HL60 cell precipitation, then add 12mL PBS buffer solution re-suspended cell, centrifugal 6min under 800 revs/min again, supernatant discarded obtains the HL60 cell precipitation after washing;
2, the HL60 cell precipitation after washing is suspended with cells frozen storing liquid, then carry out cell counting, and dilute with above-mentioned cells frozen storing liquid, obtaining concentration is 2 × 10
6the HL60 cell of the logarithmic phase of individual/milliliter and the mixed liquor of cells frozen storing liquid; Wherein, above-mentioned cells frozen storing liquid by volume percentage comprises 90% full cell culture fluid and 10% dimethyl sulfoxide (DMSO), and this full cell culture fluid comprises that volume ratio is cell culture medium and the hyclone of 9:1, and cell culture medium comprises following component:
Four water-calcium nitrate 100mg/L; Potassium chloride 400mg/L; Anhydrous magnesium sulfate 48.84mg/L; Sodium chloride 6000mg/L; ADSP 800mg/L; L-R-gene 200mg/L; L-asparagine 50mg/L; L-ASPARTIC ACID 20mg/L; CYSTINE hydrochloride 65.15mg/L; Pidolidone 20mg/L; Glu 300mg/L; Glycine 10mg/L; L-histidine monohydrochloride 15mg/L; L-hydroxyproline 20mg/L; ILE 50mg/L; LYS 40mg/L; METHIONINE 15mg/L; L-Phe 15mg/L; L-PROLINE 20mg/L; Serine 30mg/L; L-threonine 20mg/L; L-Trp 5mg/L; TYR 20mg/L; Valine 20mg/L; DEXTROSE ANHYDROUS 2000mg/mL; Reduced glutathione 1mg/L; Phenol red 5mg/L; Bio 0.2mg/L; Calcium pantothenate 0.25mg/L; Choline Chloride 3mg/L; Folic acid 1mg/L; Inositol 35mg/L; Vitamin PP 1mg/L; P-aminobenzoic acid 1mg/L; Cobastab
61mg/L; Cobastab
20.2mg/L; Cobastab
11mg/L and Cobastab
120.005mgL;
3, be 2 × 10 by concentration
6the HL60 cell of logarithmic phase and the mixed liquor of cells frozen storing liquid of individual/milliliter are sub-packed in the aseptic cryopreservation tube of 1mL;
4, concentration will be housed is 2 × 10
6individual/HL60 the cell of logarithmic phase of milliliter and the multiple aseptic cryopreservation tube of the mixed liquor of above-mentioned cells frozen storing liquid are positioned over program temperature reduction box (Nalgene, article No.: 5100-0001C) in, at-80 DEG C, place 48 hours, then multiple aseptic cryopreservation tubes are transferred in liquid nitrogen and are preserved.
After frozen 6 months, carry out cell recovery, first from liquid nitrogen, take out 1 aseptic cryopreservation tube, this aseptic cryopreservation tube is put into 37 DEG C of water-baths immediately, shake was melted freeze-stored cell in 2 minutes; Then under 800 revs/min centrifugal 5 minutes, abandon supernatant; Add the above-mentioned full cell culture fluid re-suspended cell of 1ml, this cell and the suspension of full cell culture fluid are added in the above-mentioned full cell culture fluid of 10ml, at 37 DEG C, leave standstill and cultivate 20 hours, measure the survival rate of cell, survival rate is 88%.
The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. a cells frozen storing liquid, is characterized in that, by volume percentage comprises following component:
Full cell culture fluid 90 ~ 95%;
Dimethyl sulfoxide (DMSO) 5 ~ 10%;
Wherein, described full cell culture fluid comprises that volume ratio is cell culture medium and the hyclone of 9:1.
2. cells frozen storing liquid according to claim 1, is characterized in that, by volume percentage comprises following component:
Full cell culture fluid 90%;
Dimethyl sulfoxide (DMSO) 10%;
Wherein, described full cell culture fluid comprises that volume ratio is cell culture medium and the hyclone of 9:1.
3. cells frozen storing liquid according to claim 1, is characterized in that, described cell culture medium comprises following component:
Anhydrous calcium chloride 265.00mg/L; Ferric nitrate 0.10mg/L; Potassium chloride 400.00mg/L; Anhydrous magnesium sulfate 97.67mg/L; Sodium chloride 6400.00mg/L; AMSP 109.00mg/L; Succinic acid 75.00mg/L; Sodium succinate 100.00mg/L; L-R-gene 84.00mg/L; L-hydrochloric acid cystine 63.00mg/L; Glycine 30.00mg/L; L-histidine monohydrochloride 42.00mg/L; ILE 105.00mg/L; L-Leu 105.00mg/L; LYS 146.00mg/L; METHIONINE 30.00mg/L; L-Phe 66.00mg/L; Serine 42.00mg/L; L-threonine 95.00mg/L; L-Trp 16.00mg/L; TYR 72.00mg/L; Valine 94.00mg/L; D-VB5 calcium 4.00mg/L; Choline tartrate 7.20mg/L; Folic acid mg/L; Inositol 7.20mg/L; Vitamin PP 4.00mg/L; Vitamin b3 0.40mg/L; Thiamine hydrochloride 4.00mg/L; Pyridoxine hydrochloride 4.00mg/L; Glucose 1000.00mg/L; Sodium Pyruvate 110.00mg/L and phenol red 9.30mg/L.
4. cells frozen storing liquid according to claim 1, is characterized in that, described cell culture medium comprises following component:
Calcium chloride 116.6mg/L; Copper sulphate 0.0013mg/L; Fe(NO3)39H2O 0.05mg/L; Green vitriol 0.417mg/L; Potassium chloride 311.8mg/L; Magnesium chloride 28.64mg/L; Anhydrous magnesium sulfate 48.84mg/L; Sodium chloride 6999.6mg/L; AMSP 54.35mg/L; ADSP 71.02mg/L; Zinc vitriol 0.432mg/L; L-R-gene 147.5mg/L; CYSTINE hydrochloride 31.29mg/L; Glu 365mg/L; Glycine 18.75mg/L; L-histidine monohydrochloride 31.48mg/L; ILE 54.47mg/L; L-Leu 59.05mg/L; LYS 91.25mg/L; METHIONINE 17.24mg/L; L-Phe 35.48mg/L; Serine 26.25mg/L; L-threonine 53.45mg/L; ALANINE 4.45mg/L; L-asparagine 7.5mg/L; L-ASPARTIC ACID 6.65mg/L; L-cysteine hydrochloride 17.56mg/L; Pidolidone 7.35mg/L; L-PROLINE 17.25mg/L; L-Trp 9.02mg/L; TYR mg/L; Valine 52.85mg/L; DEXTROSE ANHYDROUS 3151mg/L; Hypoxanthine 2mg/L; Linoleic acid 0.042mg/L; Lipoic acid 0.105mg/L; Phenol red 8.1mg/L; Putriscine dihydrochloride 0.081mg/L; Sodium Pyruvate 55mg/L; Biotin 0.0035mg/L; D-VB5 calcium 2.24mg/L; Choline Chloride 8.98mg/L; Folic acid 2.65mg/L; Inositol 12.6mg/L; Vitamin PP 2.02mg/L; Pyridoxal hydrochloride 2mg/L; Puridoxine hydrochloride 0.03mgL; Vitamin b3 0.219mg/L; Thiamine hydrochloride 2.17mg/L; Thymidine 0.365mg/L and Cobastab
120.68mg/L.
5. cells frozen storing liquid according to claim 1, is characterized in that, described cell culture medium comprises following component:
Four water-calcium nitrate 100mg/L; Potassium chloride 400mg/L; Anhydrous magnesium sulfate 48.84mg/L; Sodium chloride 6000mg/L; ADSP 800mg/L; L-R-gene 200mg/L; L-asparagine 50mg/L; L-ASPARTIC ACID 20mg/L; CYSTINE hydrochloride 65.15mg/L; Pidolidone 20mg/L; Glu 300mg/L; Glycine 10mg/L; L-histidine monohydrochloride 15mg/L; L-hydroxyproline 20mg/L; ILE 50mg/L; LYS 40mg/L; METHIONINE 15mg/L; L-Phe 15mg/L; L-PROLINE 20mg/L; Serine 30mg/L; L-threonine 20mg/L; L-Trp 5mg/L; TYR 20mg/L; Valine 20mg/L; DEXTROSE ANHYDROUS 2000mg/mL; Reduced glutathione 1mg/L; Phenol red 5mg/L; Bio 0.2mg/L; Calcium pantothenate 0.25mg/L; Choline Chloride 3mg/L; Folic acid 1mg/L; Inositol 35mg/L; Vitamin PP 1mg/L; P-aminobenzoic acid 1mg/L; Cobastab
61mg/L; Cobastab
20.2mg/L; Cobastab
11mg/L and Cobastab
120.005mg/L.
6. a cell freezing method, is characterized in that, comprises the steps:
The cell of logarithmic phase is washed with PBS buffer solution, and the cell of the logarithmic phase after described washing is suspended with the cells frozen storing liquid as described in claim 1 ~ 5 any one, then carry out cell counting, and dilute with described cells frozen storing liquid, obtaining concentration is 0.5 ~ 5 × 10
6the cell of logarithmic phase and the mixed liquor of described cells frozen storing liquid of individual/milliliter;
Be 0.5 ~ 5 × 10 by described concentration
6the cell of logarithmic phase and the mixed liquor of described cells frozen storing liquid of individual/milliliter are sub-packed in the aseptic cryopreservation tube of 1mL;
Described in inciting somebody to action, concentration being housed is 0.5 ~ 5 × 10
6individual/the cell of logarithmic phase of milliliter and the aseptic cryopreservation tube of the mixed liquor of described cells frozen storing liquid are positioned in program temperature reduction box and are cooled to-80 DEG C, and at-80 DEG C, place 24 hours ~ 96 hours, then described aseptic cryopreservation tube are transferred in liquid nitrogen and are preserved.
7. cell freezing method according to claim 6, it is characterized in that, described cell is attached cell, the described step that the cell of logarithmic phase is washed with PBS buffer solution is: described attached cell is cultured to after logarithmic phase, remove the cell culture fluid in culture dish, add PBS buffer solution to wash, then remove described PBS buffer solution.
8. cell freezing method according to claim 7, it is characterized in that, after the step that the cell of logarithmic phase is washed with PBS buffer solution, also comprise the step of cell dissociation, the step of described cell dissociation is: adding 0.5 ~ 2mL concentration to the surface of described attached cell is 0.25% trypsin solution, in 37 DEG C of incubators, digest 2 ~ 5 minutes, add again the full cell culture fluid of cells frozen storing liquid described in 3 ~ 5mL, to stop described tryptic digestion, then described attached cell is dispelled, and by the mixed liquor of described attached cell and described full cell culture fluid under 500 revs/min ~ 1000 revs/min centrifugal 3 minutes ~ 10 minutes, supernatant discarded obtains cell precipitation.
9. cell freezing method according to claim 8, is characterized in that, contains concentration and be 0.38% ethylenediamine tetra-acetic acid in described trypsin solution.
10. cell freezing method according to claim 6, it is characterized in that, described cell is suspension cell, described the cell of logarithmic phase is specially by the step that PBS buffer solution washs: by described suspension cell culture to logarithmic phase, by the cell culture fluid of the cell that contains logarithmic phase under 500 revs/min ~ 1000 revs/min centrifugal 3 minutes ~ 10 minutes, supernatant discarded obtains cell precipitation, add PBS buffer solution, under 500 revs/min ~ 1000 revs/min centrifugal 3 minutes ~ 10 minutes again, supernatant discarded obtained the cell precipitation after washing.
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| WO2023273046A1 (en) * | 2021-06-30 | 2023-01-05 | 苏州大学 | Cell cryopreservation solution with egg white, and preparation method therefor and use thereof |
| CN115247150A (en) * | 2021-12-17 | 2022-10-28 | 黑龙江诺亚康大干细胞技术有限责任公司 | Culture and cryopreservation method of NK (natural killer) cells |
| CN114698629A (en) * | 2022-05-11 | 2022-07-05 | 江苏真旺农业生物科技有限公司 | Cell storage device and cell storage method for optimizing cell preservation |
| CN114698629B (en) * | 2022-05-11 | 2023-08-25 | 黑龙江国智生物工程有限公司 | Cell storage device and cell storage method for optimizing cell preservation |
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