CN104911147A - Serum-free medium and preparation method thereof - Google Patents
Serum-free medium and preparation method thereof Download PDFInfo
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- CN104911147A CN104911147A CN201510383491.7A CN201510383491A CN104911147A CN 104911147 A CN104911147 A CN 104911147A CN 201510383491 A CN201510383491 A CN 201510383491A CN 104911147 A CN104911147 A CN 104911147A
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Abstract
The invention relates to a serum-free medium and a preparation method thereof. The preparation method of the serum-free medium includes: loading fatty acid on FAF (fatty-acid-free) albumin, and then adding an obtained albumin solution into a basic culture medium. The serum-free medium obtained by using the preparation method of the serum-free medium is stable in component and good in functional consistency, and no difference can be caused due to difference of species origins or processing batches of the albumin.
Description
Technical field
The present invention relates to a kind of serum free medium and compound method thereof, belong to technical field of cell culture.
Background technology
Serum free medium refers to not to be needed to add the synthetic medium that serum just can maintain cell long period growth and breeding in vitro, cultivation immunocyte can be applied to, such as lymphocyte (such as, T lymphocyte, B cell, NK cell etc.), dendritic cell (also claiming DC cell), Monocytes/Macrophages etc.
As a rule, immunocyte is survived and nutritive substance required for increasing comprises: albumin, Transferrins,iron complexes, Regular Insulin, amino acid, VITAMIN, sugar, lipid, inorganic salt, somatomedin etc. in vitro.In these compositions, having the greatest impact of albumin cell growth and survival is also the most complicated composition of function and composition.And in serum free medium, the compositions such as the lipid acid of the function that albumin plays mainly entrained by it and trace element determine.
The albumin of different sources, the bovine albumin of such as different genera and human albumin, the lipid acid entrained by them is widely different; Such as, recombinant albumin is different from the lipid acid entrained by human serum albumin; Such as in recombinant albumin, it is different from the lipid acid entrained by the recombinant albumin that saccharomycetes to make fermentation is produced that paddy endosperm extracts; Different sources, also there is difference.In addition, later stage process of manufacture also can cause the lipid acid difference entrained by albumin, and the lipid acid entrained by albumin of that such as different manufacturers produce, different batches is also very different.In a word, these differences above just cause the complicated component, the instability that with the addition of albuminous serum free medium.
The albumin of adding in serum free medium, the lipid acid entrained by it by after cellular uptake, for building membrane structure, signal transduction molecule, changing into energy or participating in the synthesis etc. of other material.Therefore, the difference of the lipid acid entrained by the albumin in substratum can have influence on all respects such as growing multiplication, cell function of immunocyte.Particularly, often too high or too low propagation and the killing activity thereof that also can be unfavorable for immunocyte of some fatty acid content, the linolenic acid of such as too high amount or the linolic acid of crossing low levels to immune cell propagation and killing activity unfavorable.
Particularly, for lymphocyte, ex vivo enrichment, activation, and amplifying lymphocyte, can be used for the disease of the multiple such as malignant tumour of adoptive therapy, infection, autoimmune disease.But, in the process increased in vitro, need the substratum using suitable external support lymphocyte survival and amplification.
At present, certain references disclose some serum free mediums, such as, be applicable to the serum free medium cultivating T lymphocyte and DC cell, but all not open solution for albuminous composition complicacy.
Summary of the invention
In view of the problems referred to above and/or the other problems of correlation technique, the invention provides the more stable serum free medium of a kind of composition and compound method thereof, improve the situation of the medium component instability caused because of albuminous composition complicacy.
One aspect of the present invention provides a kind of compound method of serum free medium, described serum free medium comprises basic medium and is applicable to the serum substitute of described serum free medium, described serum substitute comprises albumin, human transferrin, Regular Insulin and cholesterol, this compound method comprises the following steps: step one, obtains FAF albumin; Step 2, loads to lipid acid in described FAF albumin to obtain albumin solution; Step 3, preparation serum free medium: by albumin solution that step 2 obtains, and other serum substitutes except albumin are dissolved in described basic medium, to obtain serum free medium.
Preferably, in described step 2, specific lipid acid is loaded in described FAF albumin; Described specific lipid acid comprises palmitinic acid and/or stearic acid, oleic acid and linolic acid; In described step 2, palmitinic acid and/or the stearic acid of 0.1-2mmol is added according to every 1mmol FAF albumin, the oleic acid of 0.1-2mmol and the linoleic ratio of 0.1-2mmol, described specific lipid acid is added in the albuminous solution of described FAF and mixes, to obtain albumin solution.
Preferably, described specific lipid acid also comprises arachidonic acid, and the ratio of described arachidonic acid and described linoleic addition is 1:6 ~ 1:1.
Preferably, described specific lipid acid comprises palmitinic acid and stearic acid, oleic acid and linolic acid, and the ratio of the addition of described stearic acid and described palmitinic acid is 1:6 ~ 1:1.
Preferably, in step 2, after described specific lipid acid anhydrous alcohol solution, then add the albuminous solution of described FAF to, under the condition of 0-37.5 DEG C degree Celsius, be uniformly mixed and reach more than 4 hours.
Preferably, in step 3, add the albuminous ratio of 0.015 ~ 0.15mmol according to every 1L basic medium, albumin solution step 2 obtained adds in basic medium.
Preferably, the method for resin absorption or charcoal absorption is adopted to remove, lipid acid entrained in albumin to obtain FAF albumin.
Preferably, described albumin is bovine serum albumin, human serum albumin or recombinant human serum albumin.
Preferably, described recombinant human serum albumin is the recombinant human serum albumin of paddy endosperm expression or the recombinant human serum albumin of Recombinant yeast expression.
Wherein, plant-sourced recombinant human serum albumin refers to the human serum albumin utilizing gene recombination technology to express in plant, such as, and the recombinant human serum albumin (OsrHSA) that Wuhan Heyuan Biology Science and Technology Co., Ltd produces; The recombinant human serum albumin that Recombinant yeast is expressed refers to the human serum albumin utilizing gene recombination technology to express in yeast, such as, and the recombinant human serum albumin that North China Pharmaceutical Group Company Ltd produces.
Preferably, described basic medium is IMDM substratum, and with the volume of basic medium for benchmark, the addition of described human transferrin is 1 ~ 100mg/L, and the addition of described Regular Insulin is 1 ~ 100mg/L, and the addition of described cholesterol is 1 ~ 10mg/L.
Preferably, described serum substitute also comprises thanomin, and its addition is 1 ~ 10mg/L, with the volume of basic medium for benchmark.
Preferably, in step 3, the pH value of described serum free medium is adjusted between 6.8 ~ 7.5.
The present invention additionally provides a kind of serum free medium on the other hand, described serum free medium comprises basic medium and is applicable to the serum substitute of described serum free medium, described serum substitute comprises albumin, human transferrin, Regular Insulin and cholesterol, and described albumin is formed by load lipid acid in FAF albumin.
Preferably, described albumin is formed by the specific lipid acid of load in FAF albumin; Described specific lipid acid comprises palmitinic acid and/or stearic acid, oleic acid and linolic acid; Palmitinic acid and/or the stearic acid of 0.1-2mmol is added according to every 1mmol FAF albumin, the oleic acid of 0.1-2mmol and the linoleic ratio of 0.1-2mmol, add mixing in the albuminous solution of described FAF to and formed by described specific lipid acid.
Preferably, described specific lipid acid also comprises arachidonic acid, and the ratio of described arachidonic acid and described linoleic addition is 1:6 ~ 1:1.
Preferably, described specific lipid acid comprises palmitinic acid and stearic acid, oleic acid and linolic acid, and the ratio of the addition of described stearic acid and described palmitinic acid is 1:6 ~ 1:1.
Preferably, in described serum free medium, described albuminous addition is 0.015 ~ 0.15mmol/L, with the volume of basic medium for benchmark.
Preferably, described albumin is bovine serum albumin, human serum albumin or recombinant human serum albumin.
Preferably, described recombinant albumin is the recombinant human serum albumin of paddy endosperm expression or the recombinant human serum albumin of Recombinant yeast expression.
Preferably, described basic medium is IMDM substratum, and with the volume of basic medium for benchmark, the addition of described human transferrin is 1 ~ 100mg/L, and the addition of described Regular Insulin is 1 ~ 100mg/L, and the addition of described cholesterol is 1 ~ 10mg/L.
Preferably, described serum substitute also comprises thanomin, and its addition is 1 ~ 10mg/L, with the volume of basic medium for benchmark.
Preferably, the pH value of described serum free medium is 6.8 ~ 7.5.
Further aspect of the present invention additionally provides serum free medium that above-mentioned compound method prepares or above-mentioned serum free medium is cultivating the application in immunocyte.
Preferably, described immunocyte is lymphocyte or DC cell.
Preferably, described lymphocyte is T lymphocyte or NK cell.
Serum free medium compound method of the present invention, lipid acid is loaded to after in FAF albumin, again the albumin solution of the stable components of acquisition is added in basic medium, thus obtained serum-free culture based component is more stable, function consistence is better, can not be different because of the difference of albuminous Species origin or processing batch.
Accompanying drawing explanation
Fig. 1 is the result of the ability of cell proliferation of the serum free medium of embodiment 1 ~ 4;
Fig. 2 is the result of the ability of cell proliferation of the serum free medium of comparative example 1 ~ 4;
Fig. 3 is the result of the ability of cell proliferation of the serum free medium of embodiment 1 ~ 11 and comparative example 5 ~ 7;
Fig. 4 is the result of the lymph toxicity test of the serum free medium of embodiment 1 ~ 11 and comparative example 5 ~ 7;
Fig. 5 is the DC cell cultures count results of the serum free medium of embodiment 2 and comparative example 5 ~ 7.
Embodiment
The present invention is further illustrated by the following examples, but the present invention is not limited to these embodiments.
embodiment 1 ~ 4
The process for preparation of the serum free medium of embodiment 1 ~ 4 is as follows:
Step one: obtain FAF albumin;
Embodiment 1 gets bovine serum albumin (the A1933 product purchased from SIGMA company) 1.5mmol, injects and is made into 2000ml solution with water, add 50g gac, mixing; Adopt the hydrochloric acid soln adjust pH of 2mol/L to pH3.0; Be placed in 56 DEG C of water-baths 30 minutes or 4 DEG C of placements are spent the night; Centrifugal treating (4000 revs/min) removes gac; Use the NaOH solution adjust pH of 2mol/L to pH7.0 again; Last filtration sterilization obtains the FAF albumin solution of embodiment 1;
Embodiment 2 gets human serum albumin, and (purchased from the human serum albumin of Ji Lifu (Grifols) company, (product specification is 20%50ml*10g/ bottle, wherein the content of Sodium octoate is no more than 5mol/mol albumin)) 1.5mmol, specific operation process, with embodiment 1, obtains the FAF albumin solution of embodiment 2;
Embodiment 3 gets human serum albumin (purchased from human serum albumin (the product specification 20%50ml*10g/ bottle of Wuhan Institute of Biological Products Co., Ltd., wherein the content of Sodium octoate is no more than 10mol/mol albumin)) 1.5mmol, specific operation process, with embodiment 1, obtains the FAF albumin solution of embodiment 3;
Embodiment 4 is fetched water rice recombinant albumin (plant-sourced recombinant human serum albumin (lyophilized powder) product purchased from He Yuan biotech inc, Wuhan) 1.5mmol, specific operation process, with embodiment 1, obtains the FAF albumin solution of embodiment 4.
Step 2: specific lipid acid is loaded in described FAF albumin;
Get 1.2mmol palmitinic acid, 1.2mmol oleic acid and 1.2mmol linolic acid to join in 10ml dehydrated alcohol and dissolve, prepare identical 4 groups, in the FAF albumin solution that the step one joining embodiment 1-4 respectively obtains (being equivalent to the addition of 0.8mmol palmitinic acid, 0.8mmol oleic acid and 0.8mmol linolic acid in every 1mmol FAF albumin solution); Under 0-37.5 DEG C of condition, stir (100 revs/min) at least 4 hours, obtain the albumin solution of embodiment 1-4.
Step 3: preparation serum free medium;
Take IMDM culture medium powder (the I7633 product purchased from SIGMA company) and be mixed with IMDM culture medium solution with injection water, prepare identical 4 parts.
In these 4 parts of IMDM culture medium solutions, rise IMDM culture medium solution by each, the albumin solution (containing 0.0375mmol albumin) that the step 2 of adding embodiment 1-4 respectively obtains, again in every part, continue to add following material: recombinant human Transferrins,iron complexes 50mg (the T3705 product purchased from SIGMA company), recombinant human insulin 10mg (the injection of insulin liquid product purchased from Jiangsu Wanbang Biological Pharmaceutical Co., Ltd.), cholesterol 2mg (the C1231 product purchased from SIGMA company), thanomin 2mg (the E0135 product purchased from SIGMA company), xitix 2.5mg (the A4544 product purchased from SIGMA company), gentamicin sulphate 10000IU (the gentamicin sulphate injection product purchased from Shanghai No.1 Bio-Chemical Pharmacetical Industry Co., Ltd), copper sulfate 0.001mg, zinc sulfate 0.5mg and Manganous chloride tetrahydrate 0.0005mg, stirring makes abundant dissolving,
Be between 6.8-7.5 by 0.1mol/L hydrochloric acid and 0.1mol/L NaOH solution adjust ph again, 0.1um membrane filtration is degerming, packing, 4-8 degree Celsius of preservation.
embodiment 5 ~ 8
The process for preparation of the serum free medium of embodiment 5 ~ 8 and embodiment a and b is as follows:
Step one and three is with embodiment 2;
The operating process of step 2 is with embodiment 2, and difference is only the difference of the lipid acid addition in step 2;
Embodiment 5:
0.4mmol palmitinic acid, 0.4mmol oleic acid and 0.4mmol linolic acid;
Embodiment 6:
0.6mmol palmitinic acid, 0.6mmol oleic acid and 0.6mmol linolic acid;
Embodiment 7:
1.6mmol palmitinic acid, 1.6mmol oleic acid and 1.6mmol linolic acid;
Embodiment 8:
2.0mmol palmitinic acid, 2.0mmol oleic acid and 2.0mmol linolic acid;
Embodiment a
0.15mmol palmitinic acid, 0.15mmol oleic acid and 0.15mmol linolic acid;
Embodiment b
3mmol palmitinic acid, 3mmol oleic acid and 3mmol linolic acid.
In a specific embodiments of the present invention, described specific lipid acid also comprises arachidonic acid, the 1:6 ~ 1:1 of described arachidonic acid and described linoleic addition ratio.Preferably, ratio can be 1:5,1:4,1:3,1:2,2:3,3:4,4:5,5:6 etc.Preferred, ratio can be 1:3.A preferred embodiment of the present invention is see embodiment 9.
Described specific lipid acid also comprises stearic acid, and the addition ratio of described stearic acid and described palmitinic acid is 1:6 ~ 1:1.Preferably, ratio can be 1:5,1:4,1:3,1:2,2:3,3:4,4:5,5:6 etc.Preferred, ratio can be 1:2.A preferred embodiment of the present invention is see embodiment 10.
embodiment 9
The process for preparation of the serum free medium of embodiment 9 is as follows:
Step is embodiment 1 together;
In step 2, get 1.2mmol palmitinic acid, 1.2mmol oleic acid, 1.2mmol linolic acid and 0.4mmol arachidonic acid, join in 10ml dehydrated alcohol and dissolve, then join in the FAF albumin solution of step one acquisition; Under 0-37.5 DEG C of condition, stir (100 revs/min) at least 4 hours, obtain albumin solution;
In step 3, rise in IMDM culture medium solution at each, add albumin solution (containing 0.03mmol albumin) that step 2 obtains, recombinant human Transferrins,iron complexes 10mg, recombinant human insulin 20mg, cholesterol 6mg, thanomin 5mg, xitix 5mg, gentamicin sulphate 10000IU, copper sulfate 0.0005mg, zinc sulfate 0.1mg and Manganous chloride tetrahydrate 0.001mg.
embodiment 10
The process for preparation of the serum free medium of embodiment 10 is as follows:
Step is embodiment 1 together;
In step 2, get 0.8mmol palmitinic acid, 1.2mmol oleic acid, 1.2mmol linolic acid and 0.4mmol stearic acid and join in 10ml dehydrated alcohol and dissolve; Join in the FAF albumin solution of step one acquisition again; Under 0-37.5 DEG C of condition, stir (100 revs/min) at least 4 hours, obtain albumin solution;
In step 3, each rise in IMDM culture medium solution add step 2 and obtain albumin solution (containing 0.075mmol albumin), recombinant human Transferrins,iron complexes 100mg, recombinant human insulin 5mg, cholesterol 4mg, thanomin 10mg, xitix 1mg, gentamicin sulphate 10000IU, copper sulfate 0.001mg, zinc sulfate 0.5mg and Manganous chloride tetrahydrate 0.0005mg.
Embodiment 11
The process for preparation of the serum free medium of embodiment 11 is as follows:
Step is embodiment 1 together;
In step 2, get 1.2mmol stearic acid, 1.2mmol oleic acid, 1.2mmol linolic acid join in 10ml dehydrated alcohol and dissolve; Join in the FAF albumin solution of step one acquisition again; Under 0-37.5 DEG C of condition, stir (100 revs/min) at least 4 hours, obtain albumin solution;
In step 3, each rise in IMDM culture medium solution add step 2 and obtain albumin solution (containing 0.0375mmol albumin), recombinant human Transferrins,iron complexes 50mg, recombinant human insulin 10mg, cholesterol 4mg, thanomin 10mg, xitix 1mg, gentamicin sulphate 10000IU, copper sulfate 0.001mg, zinc sulfate 0.5mg and Manganous chloride tetrahydrate 0.0005mg.
Effect comparison data
Comparative example 1 ~ 4
The preparation of the serum free medium of comparative example 1 ~ 4:
Take IMDM culture medium powder injection water and be mixed with IMDM culture medium solution, prepare identical 4 parts.
In these 4 parts of IMDM culture medium solutions, rise IMDM culture medium solution by each, add the albumin of comparative example 1-4 respectively;
Comparative example 1: the aqueous solution 50ml of bovine serum albumin (the A1933 product purchased from SIGMA company), wherein containing bovine serum albumin 0.0375mmol;
Comparative example 2: ((product specification is 20%50ml*10g/ bottle to human serum albumin purchased from the human serum albumin of Ji Lifu (Grifols) company, wherein the content of Sodium octoate is no more than 5mol/mol albumin)) aqueous solution 50ml, wherein containing human serum albumin 0.0375mmol;
Comparative example 3: human serum albumin is (purchased from human serum albumin (the product specification 20%50ml*10g/ bottle of Wuhan Institute of Biological Products Co., Ltd., wherein the content of Sodium octoate is no more than 10mol/mol albumin)) aqueous solution 50ml, wherein containing human serum albumin 0.0375mmol;
Comparative example 4: the aqueous solution 50ml of paddy rice recombinant human serum albumin (plant-sourced recombinant human serum albumin (lyophilized powder) product purchased from He Yuan biotech inc, Wuhan), wherein containing paddy rice recombinant human serum albumin 0.0375mmol;
Again in every portion, continue to add following material: recombinant human Transferrins,iron complexes 50mg, recombinant human insulin 10mg, cholesterol 2mg, thanomin 2mg, xitix 2.5mg, gentamicin 10000IU, copper sulfate 0.001mg, zinc sulfate 0.5mg and Manganous chloride tetrahydrate 0.0005mg, stir and make abundant dissolving; Be between 6.8-7.5 by 0.1mol/L hydrochloric acid and 0.1mol/L NaOH solution adjust ph again, 0.1um membrane filtration is degerming, packing, 4-8 degree Celsius of preservation.
Comparative example 5 ~ 7
Comparative example 5: commercially available AIM-V substratum (the 087-0112BK product purchased from GIBCO company);
Comparative example 6: commercially available GT-T561 substratum (serum-free production medium purchased from TAKARA company);
Comparative example 7: commercially available RPMI1640 substratum (purchased from GIBCO company 22400-105 product), adds 5% hot deactivation autologous plasma.
Lymphopoiesis capacity experimental
In 6 orifice plates, according to the multiple hole of often kind of substratum three, every hole adds 2ml serum free medium, then adds 1*10
6periphery blood T lymphocyte, then add 300IU/ml IL-2 and 50ng/ml OKT3, cultivate, when cell density is greater than 2*10
6carry out fluid infusion cultivation during/ml, when fluid infusion is cultivated, cell density is adjusted to 1*10
6/ ml, adds 300IU/ml IL-2.The sample getting every hole on the 10th day carries out cell counting, and calculates the multiple of cell proliferation.
The result of the ability of cell proliferation of the serum free medium of embodiment 1 ~ 4 is see Fig. 1;
The result of the ability of cell proliferation of the serum free medium of comparative example 1 ~ 4 is see Fig. 2;
The result of the ability of cell proliferation of the serum free medium of embodiment 1-11, comparative example 5 ~ 7 is see Fig. 3.In addition, the result of embodiment a and embodiment 5 result closely, therefore only illustrate the result of embodiment 1 in Fig. 3; The result of embodiment b and the result of embodiment 8 closely, therefore only illustrate the result of embodiment 8 in Fig. 3.
Conclusion: as can be seen from the result of Fig. 2, conventionally the cell proliferation fold difference of serum free medium that is mixed with of method is very large for the albumin (bovine serum albumin, human serum albumin and paddy rice recombinant human serum albumin) in different genera source, the different manufacturers albumin (human serum albumin of comparative example 2 and 3) of producing, and namely cell cultures difference on effect is very large;
But, as can be seen from the result of Fig. 1, the albumin (human serum albumin of embodiment 2 and 3) that the albumin (bovine serum albumin, human serum albumin and paddy rice recombinant albumin) in different genera source, different manufacturers are produced, the cell cultures effect (cell proliferation multiple) of the serum free medium be mixed with through method of the present invention is in same level substantially, and difference is little.
This illustrates that the composition of the serum free medium adopting method of the present invention to obtain is more stable, and function consistence is better.
In addition, the result of comparative example 1-4 and comparative example 1-4 respectively, can find out that the ability of cell proliferation of the serum free medium of embodiment 1-4 is better than comparative example 1-4's, therefore, this illustrates the serum free medium adopting method of the present invention to obtain, and not only composition is more stable, function consistence is better, and ability of cell proliferation is improved.
The result of comparative example 1-4 and comparative example 5-7, can find out that the ability of cell proliferation of the serum free medium of embodiments of the invention 1-4 is better than the substratum of comparative example 5-7.
Lymphocytotoxicity is tested
By cultivating the lymphocyte action effect cell of the 10th day in above-mentioned ability of cell proliferation experiment, with Daudi cell for target cell.
First prepare effector cell solution: Example 1-11, and the substratum of comparative example 5-7 cultivate the T lymphocyte of 10 days, wash 2 times with IMDM substratum, be made into 1x10 with substratum
6/ ml cell suspension.Target cell daudi cell IMDM substratum is washed once, is made into 1x10
5/ ml cell suspension.Be inoculated in 96 orifice plates by effect target than 10:1, effector cell hole and Target cell wells are set simultaneously, by the multiple hole of often kind of substratum 3,37 degree of 5%CO
2cultivate 24 hours.Survey absorbance with mtt assay, calculate and kill ratio of outflow.
Kill ratio of outflow %=1-(test holes-effector cell hole)/Target cell wells.
Result is see Fig. 4.
Conclusion: as can be seen from Figure 4, the serum free medium of embodiment of the present invention 1-11 cultivate out lymphocyticly kill ratio of outflow, most of with commercially available AIM-V substratum, GT-T561 substratum and the RPMI1640 substratum containing 5% hot deactivation autologous plasma are comparatively close.
The serum free medium of the embodiment of the present invention, except for cultivating except T lymphocyte, can also be used for cultivating other lymphocytes, such as NK cell and B cell etc.Concrete substratum compound method, and cell culture processes ginseng is above-mentioned, specifically repeats no more.
Except lymphocyte, the serum free medium of the embodiment of the present invention can also be used for cultivating other immunocytes, such as DC cell; The cultural method ginseng of DC cell is following:
The cultivation of DC cell
The serum free medium that Example 2 is prepared, the serum free medium of comparative example 5 ~ 7 are as the serum free medium of DC cell.
Get peripheral blood lymphocytes (PBMC), first adjust cell density to 5*10 with RPMI1640 substratum
6/ ml, seeds cells in 6 orifice plates by every hole 2ml and (inoculates 12 holes altogether).Put into incubator again and cultivate 2 hours, take out, by rocking, non-adherent cell is split away off, and absorb substratum.The attached cell now stayed is the precursor cell monocyte of DC cell, add serum free medium again to cultivate DC cell: i.e. the serum free medium of Example 2 preparation, the serum free medium of comparative example 5 ~ 7, often kind of substratum 3 multiple holes, every hole 3ml serum free medium (in addition, also containing 100ng/ml IL-4 and 1000IU/ml GM-CSF in these serum free mediums) cultivation 5 days in 37 degree 5% CO2gas incubator, inducing culture becomes DC cell.Add TNFa according to 1000IU/ml, continue cultivation and after 2 days, obtain ripe DC cell.After 7th day observation of cell form, scraper scrapes cell, counts every porocyte number.The count results of DC cell is see Fig. 5.
Conclusion: in form, the serum free medium of the embodiment of the present invention 2 the DC cellular form of cultivating out similar to serum free mediums such as commercially available AIM-V.In cell proliferation quantity, the serum free medium that the embodiment of the present invention 2 is prepared is higher than AIM-V, GT-T561 and the RPMI1640 substratum containing 5% hot deactivation autologous plasma.
Be to be understood that, although this specification sheets is described according to embodiment, but not each embodiment only comprises an independently technical scheme, this narrating mode of specification sheets is only for clarity sake, those skilled in the art should by specification sheets integrally, technical scheme in each embodiment also through appropriately combined, can form other embodiments that it will be appreciated by those skilled in the art that.
A series of detailed description listed is above only illustrating for feasibility embodiment of the present invention; they are also not used to limit the scope of the invention, all do not depart from the skill of the present invention equivalent implementations done of spirit or change all should be included within protection scope of the present invention.
Claims (23)
1. the compound method of a serum free medium, described serum free medium comprises basic medium and is applicable to the serum substitute of described serum free medium, described serum substitute comprises albumin, human transferrin, Regular Insulin and cholesterol, it is characterized in that: this compound method comprises the following steps:
Step one, obtains FAF albumin;
Step 2, loads to lipid acid in described FAF albumin to obtain albumin solution;
Step 3, preparation serum free medium: by albumin solution that step 2 obtains, and other serum substitutes except albumin are dissolved in described basic medium, to obtain serum free medium.
2. compound method as claimed in claim 1, is characterized in that:
In described step 2, specific lipid acid is loaded in described FAF albumin;
Described specific lipid acid comprises palmitinic acid and/or stearic acid, oleic acid and linolic acid;
In described step 2, palmitinic acid and/or the stearic acid of 0.1-2mmol is added according to every 1mmol FAF albumin, the oleic acid of 0.1-2mmol and the linoleic ratio of 0.1-2mmol, described specific lipid acid is added in the albuminous solution of described FAF and mixes, to obtain albumin solution.
3. compound method as claimed in claim 2, is characterized in that:
Described specific lipid acid also comprises arachidonic acid, and the ratio of described arachidonic acid and described linoleic addition is 1:6 ~ 1:1.
4. compound method as claimed in claim 2, it is characterized in that: described specific lipid acid comprises palmitinic acid and stearic acid, oleic acid and linolic acid, the ratio of the addition of described stearic acid and described palmitinic acid is 1:6 ~ 1:1.
5. compound method as claimed in claim 1, is characterized in that: in step 2, after described lipid acid anhydrous alcohol solution, then adds the albuminous solution of described FAF to, under the condition of 0-37.5 DEG C degree Celsius, be uniformly mixed and reach more than 4 hours.
6. compound method as claimed in claim 1, is characterized in that: in step 3, and add the albuminous ratio of 0.015 ~ 0.15mmol according to every 1L basic medium, albumin solution step 2 obtained adds in basic medium.
7. as the compound method in claim 1-6 as described in any one, it is characterized in that: adopt the method for resin absorption or charcoal absorption to remove, lipid acid entrained in albumin to obtain FAF albumin.
8. as the compound method in claim 1-6 as described in any one, it is characterized in that: described albumin is bovine serum albumin, human serum albumin or recombinant human serum albumin.
9. compound method as claimed in claim 8, is characterized in that: described recombinant human serum albumin is the recombinant human serum albumin of paddy endosperm expression or the recombinant human serum albumin of Recombinant yeast expression.
10. as the compound method in claim 1-6 as described in any one, it is characterized in that: in step 3, the pH value of described serum free medium is adjusted between 6.8 ~ 7.5.
11. 1 kinds of serum free mediums, described serum free medium comprises basic medium and is applicable to the serum substitute of described serum free medium, and described serum substitute comprises albumin, human transferrin, Regular Insulin and cholesterol, it is characterized in that:
Described albumin is formed by load lipid acid in FAF albumin.
12. serum free mediums as claimed in claim 11, is characterized in that:
Described albumin is formed by the specific lipid acid of load in FAF albumin;
Described specific lipid acid comprises palmitinic acid and/or stearic acid, oleic acid and linolic acid; Palmitinic acid and/or the stearic acid of 0.1-2mmol is added according to every 1mmol FAF albumin, the oleic acid of 0.1-2mmol and the linoleic ratio of 0.1-2mmol, add mixing in the albuminous solution of described FAF to and formed by described specific lipid acid.
13. serum free mediums as claimed in claim 12, is characterized in that: described specific lipid acid also comprises arachidonic acid, the ratio of described arachidonic acid and described linoleic addition is 1:6 ~ 1:1.
14. serum free mediums as claimed in claim 12, is characterized in that: described specific lipid acid comprises palmitinic acid and stearic acid, oleic acid and linolic acid, the ratio of the addition of described stearic acid and described palmitinic acid is 1:6 ~ 1:1.
15. serum free mediums as claimed in claim 11, it is characterized in that: in described serum free medium, described albuminous addition is 0.015 ~ 0.15mmol/L, with the volume of basic medium for benchmark.
16., as the serum free medium in claim 11-15 as described in any one, is characterized in that: described albumin is bovine serum albumin, human serum albumin or recombinant human serum albumin.
17. serum free mediums as claimed in claim 16, is characterized in that: described recombinant albumin is the recombinant human serum albumin of paddy endosperm expression or the recombinant human serum albumin of Recombinant yeast expression.
18. as the serum free medium in claim 11-15 as described in any one, it is characterized in that: described basic medium is IMDM substratum, with the volume of basic medium for benchmark, the addition of described human transferrin is 1 ~ 100mg/L, the addition of described Regular Insulin is 1 ~ 100mg/L, and the addition of described cholesterol is 1 ~ 10mg/L.
19. serum free mediums as claimed in claim 18, it is characterized in that: described serum substitute also comprises thanomin, its addition is 1 ~ 10mg/L, with the volume of basic medium for benchmark.
20., as the serum free medium in claim 11-15 as described in any one, is characterized in that: the pH value of described serum free medium is 6.8 ~ 7.5.
21. as the compound method in claim 1-6 as described in any one the serum free medium prepared, or be applied to cultivation immunocyte as the serum free medium in claim 11-15 as described in any one.
22. apply as claimed in claim 21, it is characterized in that: described immunocyte is lymphocyte or DC cell.
23. apply as claimed in claim 22, it is characterized in that: described lymphocyte is T lymphocyte or NK cell.
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| CN109097331A (en) * | 2018-09-25 | 2018-12-28 | 深圳市五零生命科技有限公司 | A kind of NK cell non-serum culture medium |
| CN113980895A (en) * | 2020-07-27 | 2022-01-28 | 山东荆卫生物科技有限公司 | Improved culture medium for human umbilical cord mesenchymal stem cells and method for separating and purifying human umbilical cord mesenchymal stem cells |
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