CN105543163A - Serum-free culture medium used for full-suspension culture of MDCK (Madin Darby Canine Kidney) cells - Google Patents
Serum-free culture medium used for full-suspension culture of MDCK (Madin Darby Canine Kidney) cells Download PDFInfo
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Abstract
The invention provides a serum-free culture medium used for full-suspension culture of MDCK (Madin Darby Canine Kidney) cells. The culture medium is prepared from a DMEM/F12 culture medium containing 5g/L of NaCl and the following components: sodium stannite, recombinant human epidermal growth factors, hydrocortisone, recombinant full-chain insulin, prostaglandin E1, human transferrin, thyroxine (T3), vitamin E, cholesterol, ethanol amine, beta-mercaptoethanol, Tween-80, Hypep1510, recombinant human serum albumin ACF and mannitol. With the adoption of the culture medium provided by the invention, the full-suspension culture of the MDCK cells can be carried out very well under the condition that serum is not added; after the MDCK cells are continuously cultured for 20 generations, the average proliferation concentration of the MDCK cells is 2.308*10<6>/mL, the average cell viability is 97.4 percent and the average doubling time is 34.48 hours, so that the serum-free culture medium provides technical supports for developing influenza vaccines of cell matrixes of mammals in China.
Description
Technical field
The invention belongs to biological product technical field, be specifically related to a kind of serum free medium for full suspension culture mdck cell.
Background technology
Influenza (bird flu) is the Important Infectious Diseases of harm humans health and aviculture development, vaccine inoculation is one of main means of current flu-prevention (bird flu), conventional flow influenza vaccine chick embryo culture is produced, when the method exists that microbial contamination probability in preparation process is high, product endogenous toxic material content is high, injection side reaction is large, refuse has that environmental pollution treatment cost is high, flu outbreak breaks out, chicken embryo supply bottleneck and the processing disadvantages such as the production cycle is long, therefore develop safety, zooblast matrix influenza vaccines be imperative efficiently.
Mdck cell (Madin-Darbycaninekidneycells, MDCK) is by the renal tissue separation and Culture of Madin and Darby in 1958 from the female bent frame dog of U.S. CockerSpaniel.Be generally acknowledge at present be suitable for one of ideal cell line that first, influenza B virus produces, the MedImmune company approval under existing Dutch Su Wei drugmaker, Novartis Co., Ltd of Switzerland and U.S.'s AstraZeneca utilizes mdck cell to produce influenza vaccines.Ye You many enterprises of China are utilizing mdck cell to carry out the research and development of influenza vaccines, but the influenza vaccines listing also not having the mdck cell of independent research to produce at present, reason is except Chinese Enterprises is started late, and mdck cell cultivates the important factor that personalized substratum is also restriction China cell matrix influenza vaccines research and development process.
The many cytostromatic vaccines of China are carrying out or are completing from traditional rolling bottle technique to the upgrading and transformation of bio-reactor high-density culture technique, bioreactor culture cell has two kinds of modes, one utilizes cell itself will be attached to the epontic characteristic of culture to carry out microcarrier cultivation, when this mode will realize large scale culturing, operational path complexity is not easy to grasp, the result caused cultivates small scale under existence conditions, the small-sized biological reactor ability production task wanting usage quantity a lot of aborning, because the difference between reactor finally causes whole product to have larger difference between batch, and the anchoring factor that animal serum will be used in culture process to provide cell to attach on microcarrier, add risk and the downstream purification technique of Biosafety.Cell is tamed into and can be carried out the full suspension culture of bio-reactor after the cell of suspension culture entirely by the second, current BHK21 cell all completes technique upgrading in aftosa vaccine, single tank volume of culture of domestic bio-reactor serum-free culture can reach 3000L, is 60 times more than that microcarrier cultivates single tank volume.The easy linear amplification of full suspension culture technique, but also can serum free medium be adopted, improve the biological safety of derived product, simplify purification work, is the direction of current novel vaccine research and development.
Serum free medium uses the serum substitute of definite ingredients, not only simplify downstream process, it also avoid biological pollution risk, but serum composition is still unclear at present, moreover mdck cell itself requires high to culture condition, develops the Focal point and difficult point that this cell non-serum culture medium is this area research always.
Summary of the invention
In order to solve problems of the prior art, the invention provides a kind of serum free medium for full suspension culture mdck cell, for China's influenza vaccines bioreactor culture technique provides support.
First object of the present invention is to provide a kind of serum free medium for full suspension culture mdck cell, and described substratum is by being that the DMEM/F12 substratum of 5g/L and following component form: sodium stannite containing NaCl; Recombinant human epidermal growth factor (EGF); Hydrocortisone; To recombinate full chain Regular Insulin; Prostaglandin(PG) (E1); Human transferrin; Thyroxine (T3); Vitamin-E; Cholesterol; Thanomin; Beta-mercaptoethanol; Tween-80; HyPep1510; Recombinant human serum albumin ACF; N.F,USP MANNITOL sugar.
As preferably, described substratum is by being that the DMEM/F12 substratum of 5g/L and following component form containing NaCl:
Trace element:
Sodium stannite 0.000001-0.00001g/L
Somatomedin:
Recombinant human epidermal growth factor (EGF) 0.00005-0.0005g/L
Hydrocortisone 0.000012-0.00006g/L
Recombinate full chain Regular Insulin 0.001-0.01g/L
Prostaglandin(PG) (E1) 0.00001-0.00005g/L
Human transferrin 0.002-0.008g/L
Thyroxine (T3) 1-5 × 10
-10g/L
Composite of lipid:
Vitamin-E 0.00001-0.0001g/L
Cholesterol 0.00001-0.0001g/L
Thanomin 0.0005-0.001g/L
Beta-mercaptoethanol 0.0005-0.001g/L
Tween-80 0.005-0.01g/L
Plant hydrolyzed thing:
HyPep15101-10g/L
Recombinant human serum albumin:
ACF0.5-5g/L
Other:
N.F,USP MANNITOL sugar 0.5-5g/L.
As further preferably, described substratum is by being that the DMEM/F12 substratum of 5g/L and following component form containing NaCl:
Trace element:
Sodium stannite 0.000005g/L
Somatomedin:
Recombinant human epidermal growth factor (EGF) 0.0001g/L
Hydrocortisone 0.000036g/L
Recombinate full chain Regular Insulin 0.005g/L
Prostaglandin E1 0.000025g/L
Human transferrin 0.0047g/L
Thyroxine (T3) 4.0 × 10
-10g/L
Composite of lipid:
Vitamin-E 0.00005g/L
Cholesterol 0.00005g/L
Thanomin 0.000925g/L
Beta-mercaptoethanol 0.000975g/L
Tween-80 0.00625g/L
Plant hydrolyzed thing:
HyPep15105g/L
Recombinant human serum albumin:
ACF0.5-1g/L
Other:
N.F,USP MANNITOL sugar 1g/L.
As preferably, the concentration of described recombinant human serum albumin ACF is 0.5g/L or 1g/L.
Second object of the present invention is to provide the preparation method of above-mentioned substratum, it is characterized in that: be mix after fat-soluble emulsifying raw material freeze-drying with other raw material, levigate and get final product.
During use, dissolve with water for injection, often liter adds 2200mgNaHCO
3, constant volume, with 1N sodium hydroxide or 1N hydrochloric acid adjust pH to 7.1-7.2, filtration sterilization and get final product.
3rd object of the present invention is to provide the preparation method of above-mentioned substratum, it is characterized in that: by (do not need freeze-drying) after fat-soluble emulsifying raw material with other raw material be dissolved in water for injection successively, then often liter adds 2200mgNaHCO
3, constant volume, finally uses 1N sodium hydroxide or 1N hydrochloric acid adjust pH to 7.1-7.2, filtration sterilization and get final product.
4th object of the present invention is to provide the application of above-mentioned substratum in the full suspension culture mdck cell of serum-free.
5th object of the present invention is to provide the method for the full suspension culture mdck cell of the above-mentioned substratum of application, and in the arbitrary described substratum of claim 1-4, directly inoculate mdck cell, do not add serum, at 37 DEG C, under the rotating speed of 120rpm, shaking table is cultivated.
In order to obtain full suspension culture mdck cell serum free medium, the applicant has carried out great many of experiments, after substantially determining the every component contained in substratum, has carried out great many of experiments again to optimum proportioning, is below part Experiment process and experimental result.
The applicant is based on commercial DMEM/F12 culture medium prescription, regulate osmotic pressure by adjustment sodium chloride concentration and add plant protein hydrolysate, recombinant human serum albumin, somatomedin and lipid, designing and optimize the serum-free culture based formulas of MDCK suspension cell growth.
1. trial test one: directly add following additive (unit: g/L) at DMEM/F12 substratum:
Trace element:
Sodium stannite (Na
2seO
3-5H
2o) 0.000005
Somatomedin:
Recombinant human epidermal growth factor (EGF) 0.0001
Hydrocortisone 0.000036
To recombinate full chain Regular Insulin 0.005
Prostaglandin E1 0.000025
Human transferrin 0.0047
Thyroxine (T3) 4.0 × 10
-10
Composite of lipid:
Vitamin-E 0.0002
Cholesterol 0.0002
Thanomin 0.0037
Beta-mercaptoethanol 0.0039
Tween-80 0.025 (density is 1.09g/ml, by 1g/ml during weighing)
Plant hydrolyzed thing:
HyPep151010
Recombinant human serum albumin:
ACF1
Other:
N.F,USP MANNITOL sugar 1
Adjust pH to be 7.10 ± 0.2, measure osmotic pressure, after filtration sterilization, with 3.0 × 10
5the cell density inoculation MDCK suspension cell of/ml, suspension culture test cell growth result, culture temperature: 37 DEG C, rotating speed: 120rpm.
Result: the volumetric molar concentration of the substratum osmotic pressure that trial test one is prepared is 368mmol/kg, osmotic pressure molar density exceeds the scope that mdck cell bears, and during with this culture medium culturing MDCK suspension cell, cell volume significantly reduces, poor growth, can not normal growth.The results are shown in Table 1.
The result of the substratum suspension culture MDCK substratum that table 1 trial test one is prepared
2. trial test two: with DMEM/F12 formulation data for reference, except NaCl adds 5g/L, other all substances add by the standard recipe of DMEM/F12 substratum, add the identical trace element of same trial test one, somatomedin, composite of lipid, plant hydrolyzed thing, recombinant human serum albumin and N.F,USP MANNITOL sugar in addition again, pH is adjusted to be 7.10 ± 0.2, measure osmotic pressure, after filtration sterilization, with 3.0 × 10
5/ ml cell density inoculation MDCK suspension cell, carries out suspension culture test cell growth result, culture temperature: 37 DEG C, rotating speed: 120rpm.
Result: the substratum osmotic pressure molar density that trial test two is prepared is 312mmol/kg, cultivate MDCK suspension cell, single celled epidemic situation comparison is good, circle and bright, but has serious cell conglomeration, still can not normally cultivate MDCK suspension cell.The results are shown in Table 2.
The result of the substratum suspension culture MDCK substratum that table 2 trial test two is prepared
By above twice trial test all fall flats, therefore select to carry out single factor test the best cultivation concentration screening to the main additive such as somatomedin, composite of lipid, plant hydrolyzed thing and recombinant human serum albumin.
3. plant protein hydrolysate the best cultivates concentration screening
With DMEM/F12 formulation data for reference, except NaCl adds 5g/L, the standard recipe that other all substances press DMEM/F12 substratum adds, and adds sodium stannite (Na in addition
2seO
3-5H
2o) fill a prescription based on 0.000005g/L and N.F,USP MANNITOL sugar 1g/L, add 10g/L, 7.5g/L, 5g/L, 2.5g/L plant protein hydrolysate Hypep1510 respectively and carry out cultivation test, using DMEM/F12 medium base formula as blank.
Result: HyPep1510 is to the exercising result of MDCK suspension cell growth see Fig. 1, and the growth of HyPep1510 to MDCK suspension cell has obvious promoter action as can be seen from Figure 1.When to add concentration be 10g/L there is the serious group's of connecing phenomenon in cell, is cultured to the later stage particularly serious, and be cultured to 72h cell viability and obviously decline, and drops to 65% by 96.5%; When interpolation concentration is 7.5g/L there is comparatively serious conglomeration in cell, but comparatively 10g/L is good; When interpolation concentration is 5g/L there is the slightly group's of connecing phenomenon in cell; The almost acellular group of connecing when to add concentration be 2.5g/L, and cell becomes clear, size is homogeneous, and state is better.7.5g/L, 5g/L, 2.5g/L Cell viability is all more than 90%.Comprehensive Growth of Cells effect and Productive statistics cost, when adding separately, the optimum concn of HyPep1510 is defined as 2.5g/L.
4. recombinant human serum albumin the best cultivates concentration screening
With DMEM/F12 formulation data for reference, except NaCl adds 5g/L, the standard recipe that other all substances press DMEM/F12 substratum adds, and adds sodium stannite (Na in addition
2seO
3-5H
2o) fill a prescription based on 0.0000055g/L and N.F,USP MANNITOL sugar 15g/L, add 1g/L, 0.75g/L, 0.5g/L, 0.25g/L recombinant human serum albumin ACF respectively and carry out cultivation test, using DMEM/F12 medium base formula as blank.
Result: recombinant human serum albumin to the exercising result of MDCK suspension cell growth see Fig. 2, as can be seen from Figure 2,1g/L, 0.75g/L, 0.5g/L, 0.25g/L4 gradient cellular form is all better, cell size is homogeneous and do not occur that cell connects a phenomenon, visible recombinant human serum albumin ACF has important effect to maintenance MDCK suspension cell normal morphology, can infer that ACF is not the important factor causing cell conglomeration simultaneously.As seen from Figure 2, when interpolation concentration is 0.25g/L, being cultured to 72h cell density is 63 × 10
4/ mL, more much lower than 1g/L, 0.75g/L, 0.5g/L cell density.Therefore, when adding separately, the optimum concn of recombinant human serum albumin ACF is 0.5g/L.
5. growth factor mixture the best cultivates concentration screening
With DMEM/F12 formulation data for reference, except NaCl adds 5g/L, the standard recipe that other all substances press DMEM/F12 substratum adds, and adds sodium stannite (Na in addition
2seO
3-5H
2o) fill a prescription based on 0.000005g/L and N.F,USP MANNITOL sugar 1g/L, growth factor mixture is 100% calculating (unit: g/L) by following concentration combination, cultivation test is carried out, using DMEM/F12 medium base formula as blank with 100%, 75%, 50% and 25%.
Recombinant human epidermal growth factor (EGF) 0.0001
Hydrocortisone 0.000036
To recombinate full chain Regular Insulin 0.005
Prostaglandin E1 0.000025
Human transferrin 0.0047
Thyroxine (T3) 4.0 × 10
-10
Result: growth factor mixture is to the exercising result of MDCK suspension cell growth see Fig. 3, and the growth of growth factor mixture to MDCK suspension cell does not have obvious promoter action as can be seen from Figure 3, but maintaining growth to cell has important effect.When adding concentration and being respectively 100%, 75%, 50%, 25%, 100%, 75% presents cell growth inhibiting, and cell is without remarkable growth; 50% Growth of Cells is better, and in this group, 4 concentration all do not occur that cell connects a phenomenon.
6. composite of lipid the best cultivates concentration screening
With DMEM/F12 formulation data for reference, except NaCl adds 5g/L, the standard recipe that other all substances press DMEM/F12 substratum adds, and adds sodium stannite (Na in addition
2seO
3-5H
2o) fill a prescription based on 0.000005g/L and N.F,USP MANNITOL sugar 1g/L, composite of lipid is 100% calculating (unit: g/L) by following concentration combination, cultivation test is carried out, using DMEM/F12 medium base formula as blank with 100%, 75%, 50% and 25%.
Vitamin-E 0.0002
Cholesterol 0.0002
Thanomin 0.0037
Mercaptoethanol 0.0039
Tween-80 0.025
Result: composite of lipid is to the exercising result of MDCK suspension cell growth see Fig. 4, and as can be seen from Figure 4,25% group is Growth of Cells the best in this group.In culturing process, 100% group of dead cell is more, occurs a lot of black dregs in substratum, and 50%, 25% group of cellular form is good.Therefore the interpolation concentration of composite of lipid can not be too high, too high meeting causes necrocytosis, when current formulating of recipe addition, the mixture additive of interpolation 25% can reach the best effect of cell cultures, and composite of lipid also can not cause cell conglomeration.
7. single factor test the best cultivates concentration formula test
According to 3.-test-results 6., with DMEM/F12 formulation data for reference, except NaCl adds 5g/L, the standard recipe that other all substances press DMEM/F12 substratum adds, and adds sodium stannite (Na in addition
2seO
3-5H
2o) fill a prescription based on 0.000005g/L and N.F,USP MANNITOL sugar 1g/L, add again step 3.-each component optimum concn of 6. screening, namely add plant protein hydrolysate Hypep15102.5g/L, recombinant human serum albumin 0.5g/L, growth factor mixture GF50% (recombinant human epidermal growth factor (EGF) 0.00005g/L, hydrocortisone 0.000018g/L, recombinate full chain Regular Insulin 0.0025g/L, prostaglandin E1 0.0000125g/L, human transferrin 0.00235 and thyroxine T32.0 × 10
-10and composite of lipid Lip25% (vitamin-E 0.00005g/L, cholesterol 0.00005g/L, thanomin 0.000925, beta-mercaptoethanol 0.000975g/L and tween-80 0.00625g/L) g/L), be mixed with substratum to test, using basic components as blank.
Result: according to single factor test the best cultivate concentration formula Hypep1510-2.5g/L, ACF-0.5g/L, GF-50%, Lip-25% be optimum addition prepare substratum time, cell counts is see table 3, and Growth of Cells is unsatisfactory.
Table 3 cultivates the culture effect of substratum prepared by concentration formula according to single factor test the best
8. formulation optimization
On the basis of single factor experiment in early stage, with DMEM/F12 formulation data for reference, except NaCl adds 5g/L, other all substances press the standard recipe of DMEM/F12 substratum, add sodium stannite (Na in addition
2seO
3-5H
2o) fill a prescription based on 0.000005g/L and N.F,USP MANNITOL sugar 1g/L, plant protein hydrolysate HyPep1510, recombinant human serum albumin ACF, somatomedin (GF) and composite of lipid (Lip) are optimized, adopt L
16(4
5) orthogonal test, testing program is in table 4.Still fill a prescription as contrast with DMEM/F12 medium base simultaneously, carry out cell cultures determination best of breed.MDCK suspension cell inoculum density is 1.5 × 10
5individual/mL tests.
Table 4 orthogonal test L
16(4
5) level of factor
Orthogonal experiment results is see table 5.
Table 5 Orthogonal experiment results
9. the combination formula of orthogonal experiments
According to the orthogonal experiments of table 5, carry out cell cultures screening with doubling time A3B1C1D4 and maximum propagation concentration A3B3C1D4 two combinations, MDCK suspension cell inoculum density is 50 × 10
4individual/mL tests.
Result: carry out cell cultures the selection result in table 6 and Fig. 5 with best doubling time A3B1C1D4 and maximum propagation concentration A3B3C1D4 two combinations, combination cell cultures effect the combining than A3B1C1D4 of A3B3C1D4 can be found out by table 6 and Fig. 5, application A3B3C1D4 combines the substratum prepared, and the maximum propagation concentration of mdck cell is 2.45 × 10
6/ mL, the doubling time is 34.25h.
The screening of table 6 optimal medium formula
10. end formulation
With the best of breed that maximum propagation concentration (A3B3C1D4) is index, namely with DMEM/F12 formulation data for reference, except NaCl adds 5g/L, other all substances press DMEM/F12 substratum standard recipe add, add sodium stannite (Na in addition
2seO
3-5H
2o) fill a prescription based on 0.000005g/L and N.F,USP MANNITOL sugar 1g/L, add step again and 9. screen the optimum concn combination obtained, namely add plant protein hydrolysate Hypep15105g/L, recombinant human serum albumin 0.5g/L, growth factor mixture GF100% (recombinant human epidermal growth factor (EGF) 0.0001g/L, hydrocortisone 0.000036g/L, recombinate full chain Regular Insulin 0.005g/L, prostaglandin E1 0.000025g/L, human transferrin 0.0047 and thyroxine T34.0 × 10
-10and composite of lipid Lip25% (vitamin-E 0.00005g/L, cholesterol 0.00005g/L, thanomin 0.000925g/L, beta-mercaptoethanol 0.000975g/L and tween-80 0.00625ml) g/L), be mixed with substratum to test, cultured continuously 20 generation.
Result: the continuous passage of MDCK suspension cell cultivate 20 generation maximum propagation concentration, cell viability and doubling time result be see Fig. 6-Fig. 8.From Fig. 6-Fig. 8: often all better for Growth of Cells, cell size is homogeneous, and form is consistent.20 generation average proliferation density be 2.308 × 10
6/ mL, average cell vigor is 97.4%, and mean doubling time is 34.48h.
Through experiment, substratum of the present invention, can full suspension culture mdck cell well when not adding serum, for China's influenza vaccines bioreactor culture technique provides technical support.
Accompanying drawing explanation
Accompanying drawing is used to provide a further understanding of the present invention, and forms a part for specification sheets, together with embodiments of the present invention for explaining the present invention, is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is plant protein hydrolysate best screening of cultivating concentration in the medium;
Fig. 2 is recombinant human serum albumin best screening of cultivating concentration in the medium;
Fig. 3 is growth factor mixture best screening of cultivating concentration in the medium;
Fig. 4 is composite of lipid best screening of cultivating concentration in the medium;
Fig. 5 is the screening of optimal medium formula;
Fig. 6 is the maximum propagation concentration curve applying substratum continuous passage MDCK suspension cell of the present invention;
Fig. 7 is the cell viability figure applying substratum continuous passage MDCK suspension cell of the present invention;
Fig. 8 is the doubling time figure applying substratum continuous passage MDCK suspension cell of the present invention;
Fig. 9 is application substratum continuous passage MDCK suspension cell of the present invention, cultivates cytological map during 20 generation;
Figure 10 is application culture medium culturing MDCK suspension cell of the present invention, cultivates cytological map during 72h.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is commercially available.
Material source
1 cell: MDCK suspension cell, Gansu Province's System in Animal Cell Biotechnology Technical Research Center provides.
2 starting material
Substratum and serum: DMEM/F12 (Gibco company, article No.: 12500), new-born calf serum (Lanzhou people's marine life Engineering Co., Ltd lot number: 20141028);
Amino acid: ALANINE, L-arginine hydrochloride, altheine-hydrate, L-Aspartic acid, Cys-hydrate, Pidolidone, L-glutaminate, glycine, L-Histidine hydrochloride hydrate, ILE, L-Leu, L lysine HCL, METHIONINE, L-Phe, L-PROLINE, Serine, L-threonine, L-Trp, Valine, CYSTINE-hydrochloride hydrate (all purchasing above from Solarbio company);
VITAMIN: choline chloride 60, xanthoglobulin sodium, niacinamide, pantothenic acid, hydrochloric acid putrescine, pyridoxine hydrochloride, thiamine hydrochloride, thymidine, vitamin B12, ascorbic acid phosphoric acid esters, Bio, folic acid, riboflavin (being Sigma company to produce);
Carbohydrate: D-Glucose, seminose (traditional Chinese medicines group);
Composite of lipid: cholesterol, thanomin, linolic acid, Thioctic Acid, mercaptoethanol, tween-80 (being Sigma company to produce);
Somatomedin/hormone: recombinant human epidermal growth factor EGF, hydrocortisone, full chain Regular Insulin of recombinating, prostaglandin E1, human transferrin, thyroxine (T3) (being Sigma company to produce);
Plant protein hydrolysate: HyPep1510 (production of Sheffield company);
Recombinant human serum albumin ACF (production of Sheffield company);
Other: gsh (production of Sigma company);
Chemical reagent is domestic analytical pure.
3 equipment: CO
2incubator (U.S. Thermo3111), simple microscope (Japanese Olympus, CX21), inverted phase contrast microscope (Japanese OLYMPUSCKX41+DP72), the cell counter (U.S., INNOVATIS, CASYTT), shaking table (Shanghai Zhi Cheng analytical instrument manufacturing company, ZHWY-2102C) and electronic balance (Shanghai, Mei Teletuo benefit Shanghai main office, AR2140) etc.
Embodiment 1
Serum free medium for full suspension culture mdck cell of the present invention on the basis being the DMEM/F12 substratum of 5g/L, adds following component (unit is g/L) preparation obtain:
Trace element:
Sodium stannite (Na
2seO
3-5H
2o) 0.000005
Somatomedin:
Recombinant human epidermal growth factor (EGF) 0.0001
Hydrocortisone 0.000036
To recombinate full chain Regular Insulin 0.005
Prostaglandin E1 0.000025
Human transferrin 0.0047
Thyroxine (T3) 4.0 × 10
-10
Composite of lipid:
Vitamin-E 0.00005
Cholesterol 0.00005
Thanomin 0.000925
Beta-mercaptoethanol 0.000975
Tween-80 0.00625 (density is 1.09g/ml, by 1g/ml during weighing)
Plant hydrolyzed thing:
HyPep15105
Recombinant human serum albumin:
ACF1
Other:
N.F,USP MANNITOL sugar 1.
Emulsification method: get 2.5ml tween-80 in 50ml dehydrated alcohol, be stirred well to and dissolve completely, the vitamin-E of formula ratio, cholesterol, thanomin, linolic acid, Thioctic Acid and beta-mercaptoethanol are added successively, after often kind of material dissolves completely, add another kind of material again, obtain mixtures of fat-soluble vitamins.Separately get in a beaker and add 60ml water for injection, first open magnetic stirring apparatus, slowly add 10gPF68 dry powder, add again after ensureing the dissolving completely that adds and continue to be stirred to 10g on a small quantity and dissolve completely.In dissolution process, stirring velocity is too not fast, avoids producing too many foam as far as possible.After PF68 dry powder all adds, be settled to 100ml, mix.By the mixtures of fat-soluble vitamins for preparing and PF68 ultrasonic emulsification, add in the ratio of joined dry powder after freeze-drying.
Pulvis substratum prepared by said components: by levigate and get final product after all material (freeze-drying after fat soluble materials emulsification) mixing.Dissolve with water for injection according to a conventional method during use, add NaHCO
32200mg/L, is settled to 1L, can use with after 1N sodium hydroxide or 1N hydrochloric acid adjust pH to 7.1-7.2 filtration sterilization.
Said components is prepared liquid nutrient medium: dissolved successively by above-mentioned all material, do not need freeze-drying directly to use after fat soluble materials emulsification, finally add NaHCO
32200mg/L, is settled to 1L, can use with after 1N sodium hydroxide or 1N hydrochloric acid adjust pH to 7.1-7.2 filtration sterilization.
The substratum suspension culture mdck cell of application the present embodiment, do not need to add serum, cell-seeding-density is: 3.0 × 10
5/ ml, culture temperature: 37 DEG C, rotating speed: 120rpm.Result, see Fig. 9, cultivates the cytological map in 20 generations.
Embodiment 2
The difference of the present embodiment and embodiment 1 is:
In culture medium prescription, the concentration of recombinant human serum albumin ACF is 0.5g/L.
Rest part is all identical with embodiment 1.
The substratum suspension culture mdck cell of application the present embodiment, do not need to add serum, cell-seeding-density is: 3.0 × 10
5/ ml, culture temperature: 37 DEG C, rotating speed: 120rpm.Result, see Figure 10, cultivates the cytological map of 72h.
Embodiment 3
Serum free medium for full suspension culture mdck cell of the present invention on the basis being the DMEM/F12 substratum of 5g/L, adds following component (unit is g/L) preparation obtain:
Trace element:
Sodium stannite (Na
2seO
3-5H
2o) 0.000001
Somatomedin:
Recombinant human epidermal growth factor (EGF) 0.00005
Hydrocortisone 0.000012
To recombinate full chain Regular Insulin 0.001
Prostaglandin E1 0.00001
Human transferrin 0.002
Thyroxine (T3) 1.0 × 10
-10
Composite of lipid:
Vitamin-E 0.0001
Cholesterol 0.0001
Thanomin 0.001
Beta-mercaptoethanol 0.001
Tween-80 0.01 (density is 1.09g/ml, by 1g/ml during weighing)
Plant hydrolyzed thing:
HyPep15101
Recombinant human serum albumin:
ACF0.5
Other:
N.F,USP MANNITOL sugar 5.
In the present embodiment, the preparation method of substratum is identical with embodiment 1.
Embodiment 4
Serum free medium for full suspension culture mdck cell of the present invention on the basis being the DMEM/F12 substratum of 5g/L, adds following component (unit is g/L) preparation obtain:
Trace element:
Sodium stannite (Na
2seO
3-5H
2o) 0.00001
Somatomedin:
Recombinant human epidermal growth factor (EGF) 0.0005
Hydrocortisone 0.00006
To recombinate full chain Regular Insulin 0.01
Prostaglandin E1 0.00005
Human transferrin 0.008
Thyroxine (T3) 5.0 × 10
-10
Composite of lipid:
Vitamin-E 0.00001
Cholesterol 0.00001
Thanomin 0.0005
Beta-mercaptoethanol 0.0005
Tween-80 0.005 (density is 1.09g/ml, by 1g/ml during weighing)
Plant hydrolyzed thing:
HyPep151010
Recombinant human serum albumin:
ACF5
Other:
N.F,USP MANNITOL sugar 0.5.
In the present embodiment, the preparation method of substratum is identical with embodiment 1.
Embodiment 5
Serum free medium for full suspension culture mdck cell of the present invention on the basis being the DMEM/F12 substratum of 5g/L, adds following component (unit is g/L) preparation obtain:
Trace element:
Sodium stannite (Na
2seO
3-5H
2o) 0.000003
Somatomedin:
Recombinant human epidermal growth factor (EGF) 0.0002
Hydrocortisone 0.00003
To recombinate full chain Regular Insulin 0.006
Prostaglandin E1 0.00002
Human transferrin 0.006
Thyroxine (T3) 3.0 × 10
-10
Composite of lipid:
Vitamin-E 0.00007
Cholesterol 0.00006
Thanomin 0.0008
Beta-mercaptoethanol 0.0009
Tween-80 0.007 (density is 1.09g/ml, by 1g/ml during weighing)
Plant hydrolyzed thing:
HyPep15103
Recombinant human serum albumin:
ACF2
Other:
N.F,USP MANNITOL sugar 3.
In the present embodiment, the preparation method of substratum is identical with embodiment 1.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment to invention has been detailed description, for a person skilled in the art, it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (8)
1. for a serum free medium for full suspension culture mdck cell, it is characterized in that: described substratum is by being that the DMEM/F12 substratum of 5g/L and following component form: sodium stannite containing NaCl; Recombinant human epidermal growth factor (EGF); Hydrocortisone; To recombinate full chain Regular Insulin; Prostaglandin(PG) (E1); Human transferrin; Thyroxine (T3); Vitamin-E; Cholesterol; Thanomin; Beta-mercaptoethanol; Tween-80; HyPep1510; Recombinant human serum albumin ACF; N.F,USP MANNITOL sugar.
2. substratum according to claim 1, is characterized in that: described substratum is by being that the DMEM/F12 substratum of 5g/L and following component form containing NaCl:
Trace element:
Sodium stannite 0.000001-0.00001g/L
Somatomedin:
Recombinant human epidermal growth factor (EGF) 0.00005-0.0005g/L
Hydrocortisone 0.000012-0.00006g/L
Recombinate full chain Regular Insulin 0.001-0.01g/L
Prostaglandin(PG) (E1) 0.00001-0.00005g/L
Human transferrin 0.002-0.008g/L
Thyroxine (T3) 1-5 × 10
-10g/L
Composite of lipid:
Vitamin-E 0.00001-0.0001g/L
Cholesterol 0.00001-0.0001g/L
Thanomin 0.0005-0.001g/L
Beta-mercaptoethanol 0.0005-0.001g/L
Tween-80 0.005-0.01g/L
Plant hydrolyzed thing:
HyPep15101-10g/L
Recombinant human serum albumin:
ACF0.5-5g/L
Other:
N.F,USP MANNITOL sugar 0.5-5g/L.
3. substratum according to claim 2, is characterized in that: described substratum is by being that the DMEM/F12 substratum of 5g/L and following component form containing NaCl:
Trace element:
Sodium stannite 0.000005g/L
Somatomedin:
Recombinant human epidermal growth factor (EGF) 0.0001g/L
Hydrocortisone 0.000036g/L
Recombinate full chain Regular Insulin 0.005g/L
Prostaglandin E1 0.000025g/L
Human transferrin 0.0047g/L
Thyroxine (T3) 4.0 × 10
-10g/L
Composite of lipid:
Vitamin-E 0.00005g/L
Cholesterol 0.00005g/L
Thanomin 0.000925g/L
Beta-mercaptoethanol 0.000975g/L
Tween-80 0.00625g/L
Plant hydrolyzed thing:
HyPep15105g/L
Recombinant human serum albumin:
ACF0.5-1g/L
Other:
N.F,USP MANNITOL sugar 1g/L.
4. the substratum according to Claims 2 or 3, is characterized in that: the concentration of described recombinant human serum albumin ACF is 0.5g/L or 1g/L.
5. the preparation method of the arbitrary described substratum of claim 1-4, is characterized in that: be mix after fat-soluble emulsifying raw material freeze-drying with other raw material, levigate and get final product.
6. the preparation method of the arbitrary described substratum of claim 1-4, is characterized in that: be dissolved in water for injection successively by (not needing freeze-drying) after fat-soluble emulsifying raw material with other raw material, then often liter adds 2200mgNaHCO
3, constant volume, finally uses 1N sodium hydroxide or 1N hydrochloric acid adjust pH to 7.1-7.2, filtration sterilization and get final product.
7. the arbitrary described application of substratum in the full suspension culture mdck cell of serum-free of claim 1-4.
8. application rights requires the method for the arbitrary described full suspension culture mdck cell of substratum of 1-4, it is characterized in that: in the arbitrary described substratum of claim 1-4, directly inoculate mdck cell, do not add serum, at 37 DEG C, under the rotating speed of 120rpm, shaking table is cultivated.
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