CN105462912A - Protein-free serum-free medium suitable for diploid cell culture and application - Google Patents
Protein-free serum-free medium suitable for diploid cell culture and application Download PDFInfo
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Abstract
The invention provides a protein-free serum-free medium suitable for diploid cell culture and a preparing method thereof. The medium contains calcium chloride, magnesium sulfate, potassium chloride, sodium dihydrogen phosphate, L-arginine hydrochloride, L-asparaginate, L-cystine dihydrochloride, L-histidine hydrochloride, L-leucine, L-isoleucine and the like. The medium can meet the requirement of the growth or maintenance stage of diploid cells and prevents contact with exogenous viruses possibly existing in protein and serum to reduce potential safety hazards; as the components are simple and definite, standard products can be purchased in the market, the difference between different product batches is small, and the step of purifying down-stream products is simplified; a scientific basis is provided for large-scale standard production. In addition, the protein-free serum-free medium can be applied in the growth or maintenance stage of the diploid cells.
Description
Technical field
The invention belongs to cell engineering field, relate to a kind of without the serum free medium of albumen without hydrolyzate definite ingredients, particularly a kind of be applicable to that diploid cell cultivates without the serum free medium and its preparation method and application of albumen without hydrolyzate definite ingredients.
Background technology
Supply cytotrophy and the basic substance impelling cell proliferation when cell culture medium is cultured cell in vitro, be also the living environment of Growth of Cells and breeding, be the basis of Cell culture invitro, be widely used in field of biological product, the fields such as stem-cell therapy.Be one in cell culture medium without albumen without the serum free medium of hydrolyzate definite ingredients, its composition is completely clear and definite, not containing the material of the composition such as albumen, hydrolyzate the unknown.
Preparing in vaccination process with diploid cell, having two stages to use cell culture medium, is Growth of Cells stage (cell amplification stage) and cell maintenance stage (virus amplification stage) respectively.
According to prior art, producing in the process of vaccine with diploid cell, the cell cultures amplification stage, the conventional medium of many employing 5%-10% serum-concentrations is as MEM, DMEM, or containing Regular Insulin, the serum free medium of the indefinite materials of composition such as Transferrins,iron complexes, albumin, hydrolyzate, such culture medium cost is high, derived product purifying complex, and its exogenous virus that may carry can bring serious potential safety hazard, and without albumen, the serum free medium without hydrolyzate can effectively address these problems.Although application number is the preparation method that the patent application document of CN200710129800.3 discloses a kind of diploid cell rabies vaccine and purified rabies vaccine, the cell cultures stage of this vaccine preparation method adopts serum free medium to cultivate, and the serum free medium that the method uses all has albumen to add.Application number be 201310523326.8 patent application document disclose a kind of method that serum free medium prepares cendevax, the cell cultures stage of the method adopts serum free medium, this serum-free culture based component is indefinite, and containing albumen.
In the virus amplification stage, the many employings of current domestic production contain the conventional medium of 1%-2% serum or human serum albumin.Although application number is the preparation method that the patent application document of CN200710129799.4 discloses a kind of diploid somatic cell encephalitis B vaccine, the cell cultures amplification stage in manufacture process, employ serum free medium, but the method substratum used all adds containing albumen.Application number is the preparation method that the patent application document of CN200710129800.3 discloses a kind of diploid cell rabies vaccine and purified rabies vaccine, and the serum free medium that the cell maintenance stage of the method adopts equally all has albumen to add.Application number is a kind of method that the patent application document of CN201310523326.8 discloses serum free medium and prepares cendevax, the cell maintenance stage of the method adopts serum free medium, this serum-free culture based component is indefinite, and containing albumen.
As can be seen from the above analysis, there is the problem of at least two aspects in current production technique, is need in substratum to add serum on the one hand; Be contain the indefinite components such as albumen in substratum on the other hand, add the risk of cultivation, be also unfavorable for product purification.
Summary of the invention
The object of the present invention is to provide a kind of be applicable to that diploid cell cultivates without albumen serum free medium, have without albumen, without hydrolyzate, serum-free, advantage that component is clear and definite, the amplification of diploid cell cultured cells or virus amplification can be supported.
Another object of the present invention is to provide the above-mentioned preparation method without albumen serum free medium being applicable to diploid cell and cultivating.
Another object of the present invention is to provide above-mentioned without the serum free medium of albumen without hydrolyzate definite ingredients, specifically in diploid cell cultured cells growth phase or the application in the cell maintenance stage.
In order to achieve the above object, the present invention takes following technical scheme to realize.
The invention provides a kind of be applicable to diploid cell cultivate without albumen serum free medium, be prepared from by component in table 1:
Table 1: be applicable to diploid cell cultivate without albumen serum free medium component
Invention further provides the above-mentioned preparation method without albumen serum free medium being applicable to diploid cell and cultivating, 1-10mg tween 80 is dissolved in 50ml water for injection, then 0.0005-0.15mg linolic acid is added, 0.0005-0.85mg linolenic acid, 0.0001-0.03mg arachidonic acid, 0.2-2mg vitamin-E, 0.2-0.8mg myristic acid, 0.18-0.5mg stearic acid, treat that it dissolves fully, with water for injection fluid infusion to 500ml, add 4.5-10g sodium-chlor again, 1-20g glucose, 0-6g fructose, stir to clarify, with water for injection fluid infusion to 900ml, finally add component in table 2:
Table 2: be applicable to diploid cell cultivate without the component of albumen serum free medium except tween 80, linolic acid, linolenic acid, arachidonic acid, vitamin-E, myristic acid, stearic acid, sodium-chlor, glucose, fructose
After interpolation, continue to be stirred to and dissolve completely, then use water for injection fluid infusion to 1000ml, 0.22 μm of frit is degerming, and 4 DEG C keep in Dark Place.
By the metabolic analysis to diploid cell, determine that several amino acid such as L-Asn, L-Ile, L-Leu, L-Val are quick consumption-type amino acid, increase these amino acid whose consumptions, cell growth and maintenance all have good effect.
VITAMIN is as composition indispensable in serum free medium, and it generally participates in the metabolic regulation of cell in cellular process as coenzyme or prothetic group, if vitamin H is as the moiety of some specificity hydroxylases, and the synthesis of involved in sugar metabolism and lipid acid; Folic acid is used for cell tetrahydrobiopterin synthesis folic acid, and tetrahydrofolic acid (THFA) is important one carbon unit, plays an important role in the synthesis of nucleic acid and protein; D calcium pantothenate is transformed into acyl carrier protein and coenzyme in cell, participates in the catalyzed reaction in carbohydrate, lipid, protein metabolism.The VITAMIN proportioning being applicable to diploid cell growth and maintaining is determined by application plackett-burman method of design and center combination design method etc.
Add the fructose that cell versus glucose not easily utilizes, the generation of lactic acid in metabolic process can be reduced, thus stablize pH value; Linolic acid, linolenic acid, arachidonic acid are the necessary lipid acid of mammalian cell; The glucose optimized and glutamine proportioning, effectively can control the growing amount of lactic acid and ammonia in cellular process.
In diploid cell culturing process, citric acid, ferrous sulfate and iron nitrate combinationally use and can well substitute ferritin, and its effect is better than being used alone ironic citrate or iron nitrate; Zinc sulfate can substitute the effect of Regular Insulin, promotes the synthesis of glycogen and lipid acid; Xylo-Mucine can substitute the carrier of albumin as trace element, indispensable fatty acid; Beta-mercaptoethanol then can direct stimulate cell growth, has the partial action of serum.
Above-mentioned without the serum free medium of albumen without hydrolyzate definite ingredients, for Growth of Cells stage and the cell maintenance stage of diploid cell.After adopting substratum provided by the invention, cultivate and the maintenance stage at diploid cell, all do not use serum and albumin, can be used for the production of people with vaccines such as mad dog, varicella, poliomyelitis.
Provided by the invention be applicable to diploid cell cultivate without albumen serum free medium and preparation method thereof, there is following at least one beneficial effect:
(1) prepare without the serum free medium of albumen without hydrolyzate definite ingredients, the demand that diploid cell is cultivated or maintained can not only be met, and due to without the need to adding serum, albumen and hydrolyzate etc., thus avoid and the contact of exogenous virus that may exist in albumen, serum, thus reduce potential safety hazard;
(2) because component is simple, clear and definite, all can commercially available standardised prod, between product batches, difference is little, also simplify the purification step of derived product simultaneously; For large-scale standardized production provides scientific basis;
(3) owing to not containing the indefinite additives of composition such as albumen, component is clear and definite, significantly reduces vaccine later stage separation and purification difficulty and cost, reduces anaphylaxis risk;
(4) diploid cell is cultivated with serum-free provided by the invention, protein-free medium, can obtain having blood serum medium to cultivate suitable cell density with routine, being applied to epidemic cerebrospinal meningitis B virus, rabies virus, poliovirus be when preparing, and the virus titer of acquisition and the conventional virus titer having blood serum medium to cultivate to obtain are without significant difference.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below, apparently, accompanying drawing in below describing is only some embodiments of the present invention, for those of ordinary skills, under the prerequisite not paying creative work, other embodiment and accompanying drawing thereof can also be obtained according to these accompanying drawing illustrated embodiments.
Figure 1A cultivates the cell growth curve figure of MRC-5 cell for MEM that the substratum that adopts the present embodiment to provide respectively and serum-concentration are 10%;
The cell state figure of the culture medium culturing MRC-5 cell 72h that Figure 1B provides for employing the present embodiment;
Fig. 1 C be adopt serum-concentration be 10% MEM cultivate the cell state figure of MRC-5 cell 72h;
Fig. 2 A cultivates the cell growth curve figure of 2BS cell for MEM that the substratum that adopts the present embodiment to provide respectively and serum-concentration are 10%;
The cell state figure of the culture medium culturing 2BS cell 72h that Fig. 2 B provides for employing the present embodiment;
Fig. 2 C be adopt serum-concentration be 10% MEM cultivate the cell state figure of 2BS cell 72h.
Embodiment
Carry out clear, complete description below with reference to accompanying drawing to the technical scheme of various embodiments of the present invention, obviously, described embodiment is only a part of embodiment of the present invention, instead of whole embodiments.Based on the embodiment in the present invention, other embodiments all that those of ordinary skill in the art obtain under the prerequisite not making creative work, all belong to the scope that the present invention protects.
Embodiment 1
Prepared by the present embodiment is the substratum being applicable to MRC-5 cell cultures, and each amounts of components can with reference to the data provided in table 3, and concrete steps are as follows:
Be add tween 80 in the reaction vessel of 1000ml at capacity, then add 50ml water for injection and dissolve, then add linolic acid successively, linolenic acid, arachidonic acid, vitamin-E, myristic acid, stearic acid, treats that it dissolves fully, with water for injection fluid infusion to 500ml, then adds sodium-chlor successively, glucose, fructose, stirs to clarify, and with water for injection fluid infusion to 900ml, finally adds calcium chloride successively, magnesium sulfate, Repone K, Sodium phosphate dibasic, potassium primary phosphate, L arginine hydrochloride, L-Asp, L-Asn, L-Cys-Cys dihydrochloride, L histidine monohydrochloride, L-Leu, L-Ile, glycine, L lysine hydrochloride, L-Met, L-Phe, L-Pro, L-Ser, L-Thr, L-Trp, L-Tyr disodium salt, L-Val, L ornithine hydrochloride, L citrulline, L-Glu, oxaloacetic acid, vitamin H, choline chloride 60, vitamins C, folic acid, inositol, niacinamide, para-amino benzoic acid, D calcium pantothenate, pyridoxal hydrochloride, pyridoxine hydrochloride, riboflavin, vitamin B12, Xylo-Mucine, phenol red, thanomin, L glutamine, sodium bicarbonate, zinc sulfate, copper sulfate, iron nitrate, ammonium meta-vanadate, potassiumiodide, tin chloride, single nickel salt, water glass, Thioctic Acid, vitamin-E, Sodium Selenite, cholesterol, progesterone, thymidine, myristic acid, stearic acid, Cys, magnesium chloride, Sodium propanecarboxylate, xanthoglobulin, VITAMIN B4, ferrous sulfate, putrescine, taurine, citric acid, beta-mercaptoethanol, dexamethasone, bismuth ammonium citrate, HEPES, coffic acid, paddy third dipeptides, Jing Ti/Bao Pian COBALT NITRATE CRYSTALS/FLAKES, Sodium Tetraborate, ammonium molybdate, after interpolation, continue to be stirred to and dissolve completely, then use water for injection fluid infusion to 1000ml, 0.22 μm of frit is degerming, and 4 DEG C keep in Dark Place.
Table 3: without the serum free medium component of albumen without hydrolyzate definite ingredients in embodiment 1 and embodiment 2
In order to investigate the performance of substratum in the Growth of Cells stage that the present embodiment provides, the substratum that the present embodiment is provided and serum-concentration be 10% MEM conventionally cultivate MRC-5 cell respectively.The embodiment that the present embodiment is taked is: get the MRC-5 cell of cell generation in 10-40 generation, with 0.05% trypsinase and 0.02%EDTA (ethylenediamine tetraacetic acid (EDTA)) solution, monolayer cell is digested from culturing bottle, respectively with the present embodiment substratum prepared and the MEM re-suspended cell containing 10% serum-concentration, and all with 0.6*10
6cells/ml inoculates, and adds a certain amount of corresponding substratum and is placed in 37 DEG C of cultivations, every 24h digestion counting.
Below in conjunction with cell growth curve figure and cell state figure, the substratum that the present embodiment provides is compared with the MRC-5 cell that tradition is cultivated containing blood serum medium.Figure 1A gives the cell growth curve figure of substratum and the MEM cultivation MRC-5 cell adopting the present embodiment to provide respectively, and as can be seen from the figure, during the substratum adopting this enforcement to provide, its growth velocity is faster, can more early reach maximum cell density.The cell state figure of culture medium culturing MRC-5 cell 72h that Figure 1B provides for adopting the present embodiment, Fig. 1 C be adopt concentration be 10% MEM cultivate the cell state figure of MRC-5 cell 72h.From the contrast of Figure 1B and Fig. 1 C, the substratum provided with the present embodiment conventionally cultivates MRC-5 cell, and its growth conditions is suitable with employing MEM substratum.
In order to investigate substratum that this enforcement the provides performance in the cell maintenance stage, the culture medium culturing MRC-5 cell utilizing the present embodiment to provide, then varicella virus (Oka strain VZV and rabies virus PM strain) amplification cultivation is afterwards inoculated respectively, results virus liquid, find the suitable of the virus titer that obtains and virus harvest amount and prior art conventional medium used, its virus titer is shown in Table 4.
Table 4: the culture medium culturing MRC-5 cell that embodiment provides and conventional medium cultivate the virus titer that MRC-5 cell prepares varicella virus and rabies virus
| Substratum | Varicella (Oka strain VZV) | Rabies virus (PM rabies virus strain) |
| The present embodiment substratum | 7.3 | 8.4 |
| MEM (10% serum-concentration) | 7.25 | 8.5 |
Embodiment 2
Prepared by the present embodiment is the substratum being applicable to 2BS cell cultures, and each amounts of components can with reference to the data provided in table 3, and concrete steps are in the same manner as in Example 1.
In order to investigate the performance of substratum in the Growth of Cells stage that the present embodiment provides, the substratum that the present embodiment is provided and concentration be 10% MEM cultivate 2BS cell respectively.The embodiment that the present embodiment is taked is: get the 2BS cell of cell generation in 10-40 generation, with 0.05% trypsinase and 0.02%EDTA (ethylenediamine tetraacetic acid (EDTA)) solution, monolayer cell is digested from culturing bottle, respectively with the present embodiment substratum prepared and the MEM re-suspended cell containing 10% serum-concentration, and all with 0.55*10
6cells/ml inoculates, and adds a certain amount of corresponding substratum and is placed in 37 DEG C of cultivations, every 24h digestion counting.
Below in conjunction with cell growth curve figure and cell state figure, the substratum that the present embodiment provides is compared with the 2BS cell that tradition is cultivated containing blood serum medium.Fig. 2 A gives the cell growth curve figure of substratum and the MEM cultivation 2BS cell adopting the present embodiment to provide respectively, the cell state figure of culture medium culturing 2BS cell 72h that Fig. 2 B provides for adopting the present embodiment, Fig. 2 C be adopt concentration be 10% MEM cultivate the cell state figure of MRC-5 cell 72h.From the contrast of Fig. 2 A, 2B and 2C, the substratum provided with the present embodiment conventionally cultivates 2BS cell, and its growth conditions is suitable with employing MEM substratum, and maximum cell density is suitable.
In order to investigate substratum that this enforcement the provides performance in the cell maintenance stage, the culture medium culturing 2BS cell utilizing the present embodiment to provide, then poliovirus (sabine strain II type) is inoculated, find the suitable of the virus titer that obtains and virus harvest amount and prior art conventional medium used after testing, its virus titer is shown in Table 5.
Table 5: the culture medium culturing 2BS cell that embodiment provides and conventional medium cultivate the virus titer that 2BS cell prepares poliovirus
| Substratum | Poliovirus (sabine strain II type) |
| Substratum of the present invention | 7.56 |
| MEM (10% serum-concentration) | 7.52 |
As can be seen to embodiment 1 and implement 2 analysis, be provided by the inventionly applicable to substratum that diploid cell cultivates and can substitute conventionally in prior art have blood serum medium or protein-contg serum free medium completely; Due to himself serum-free, without albumen, without hydrolyzate, definite ingredients, this substratum has unrivaled advantage, has very large application space and development space in substratum field.
Those of ordinary skill in the art will appreciate that, embodiment described here is to help reader understanding's principle of the present invention, should be understood to that protection scope of the present invention is not limited to so special statement and embodiment.Those of ordinary skill in the art can make various other various concrete distortion and combination of not departing from essence of the present invention according to these technology enlightenment disclosed by the invention, and these distortion and combination are still in protection scope of the present invention.
Claims (4)
1. be applicable to diploid cell cultivate without an albumen serum free medium, it is characterized in that, be prepared from by following component:
。
2. the preparation method of substratum according to claim 1, it is characterized in that, 1-10mg tween 80 is dissolved in 50ml water for injection, then 0.0005-0.15mg linolic acid, 0.0005-0.85mg linolenic acid, 0.0001-0.03mg arachidonic acid, 0.2-2mg vitamin-E, 0.2-0.8mg myristic acid, 0.18-0.5mg stearic acid is added, treat that it dissolves fully, with water for injection fluid infusion to 500ml, add 4.5-10g sodium-chlor, 1-20g glucose, 0-6g fructose again, stir to clarify, with water for injection fluid infusion to 900ml, finally add again:
After interpolation, continue to be stirred to and dissolve completely, then use water for injection fluid infusion to 1000ml, 0.22 μm of frit is degerming, and 4 DEG C keep in Dark Place.
3. substratum according to claim 1, diploid cell growth phase or in the cell maintenance stage as the application of substratum.
4. substratum according to claim 3, for the production of people with mad dog, varicella, Poliomyelitis Vaccine.
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| CN109106950A (en) * | 2018-09-11 | 2019-01-01 | 中国农业科学院兰州兽医研究所 | A kind of tropical theileriosis vaccine protectant and preparation method |
| CN109536435A (en) * | 2018-12-20 | 2019-03-29 | 镇江桥美科技有限公司 | A kind of Serum-free and protein-free medium |
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| CN113957042A (en) * | 2020-07-20 | 2022-01-21 | 维他利肤(北京)生物科技有限公司 | Preparation method of stem cell zero-protein culture medium and stem cell growth factor |
| WO2024004960A1 (en) | 2022-06-27 | 2024-01-04 | 武田薬品工業株式会社 | Cell culture method |
| CN115627255A (en) * | 2022-12-01 | 2023-01-20 | 天信和(苏州)生物科技有限公司 | Method for culturing human diploid cells by adopting low-serum culture medium |
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