CN105154389A - Low-serum protein-free culture medium suitable for PK-15 cell growth and preparation method of culture medium - Google Patents
Low-serum protein-free culture medium suitable for PK-15 cell growth and preparation method of culture medium Download PDFInfo
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- CN105154389A CN105154389A CN201510664918.0A CN201510664918A CN105154389A CN 105154389 A CN105154389 A CN 105154389A CN 201510664918 A CN201510664918 A CN 201510664918A CN 105154389 A CN105154389 A CN 105154389A
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Abstract
The invention belongs to the field of cell culture and particularly relates to a low-serum protein-free culture medium suitable for PK-15 cell growth and a preparation method of the culture medium. The culture medium comprises amino acid, vitamins, inorganic salt, microelements and other components. The prepared low-serum protein-free culture medium suitable for PK-15 cell growth and the preparation method of the culture medium have several advantages that the culture medium is free of protein and animal source ingredients and chemical components are clear, interference of serum for cell culture is reduced industrially, the production cost is reduced, and cell industrial production is greatly facilitated.
Description
Technical field
The invention belongs to field of cell culture, be specifically related to a kind of low serum protein-free medium being suitable for PK-15 Growth of Cells and preparation method thereof.
Background technology
PK-15 system porcine kidney cell (PigKidney), vitro culture is Epithelial attached cell, this cell is more responsive to multiple virus, as pig circular ring virus (PCV), Pestivirus suis (CSFV), pig parvoviral (PPV) etc., can be applicable to the preparation of pig circular ring virus vaccine, classical swine fever virus vaccine, swine parvovirus vaccine etc.
Pig circular ring virus (Porcinecircovirus, PCV) is at classification Shang Shu PCV-II section, Circovirus.The red corpuscle of the many animals such as PCV not aggegation ox, sheep, pig, chicken and people, its antiserum(antisera) can not suppress pig parvoviral (PPV) to erythrocytic compendency.Main manifestations is for piglet multisystem asthenia syndrome (PostweaningMultisystemicWastingSyndrome, PMWS) and pigskin after wean are scorching and nephritic syndrome (PDNS) clinically.Except PMWS, PDNS (pigskin scorching and nephrotic syndrome), PNP (Hypertrophic necrotizing pneumonia), PRDC (PRDC (porcine respiratory disease complex)), breeding difficulty, congenitally to tremble, swine disease that enteritis etc. is relevant, mortality ratio 10% ~ 30% is not etc., more serious pig farm death rate when breaking out this disease, up to 40%, causes serious financial loss to pig industry.As everyone knows, vaccine immunity is the Main Means of prevention and control Porcine circovirus desease.
Swine fever (classicalswinefever, CSF) be a kind of high degree in contact sexually transmitted disease hot in nature caused by Pestivirus suis (CSFV), there is very strong infectivity, the pig of different ages, sex and kind all can infect, and high lethality rate, its propagation will inevitably cause huge loss to social economy, 1984 OIE (OIE) listed in category-A transmissible disease, current China is also decided to be a class transmissible disease.Swine fever, except causing except septicemia, also can cause other clinical manifestation a series of, as pregnant sow miscarriage, fetus distortion, chronic trophicity consumption, lymphocytes and platelets minimizing etc.At present, world's most countries all take strict assanation, reject swine fever infected pigs, purification swinery control and eliminate swine fever.Some developed countries have eradicated swine fever by epidemic prevention and preventions, but China not yet utterly destroys, so inoculation swine Fever Vaccine is even more important, are also one of important means controlling swine fever propagation.
Traditional training method at present for PK-15 cell is adherent culture, usually in growth and maintain base, needs the animal serum adding 10%-20%, with the support albumen providing the nutritive ingredient needed for Growth of Cells and cell to keep normal morphology.This traditional technology efficiency is low, production cost is high; Significant difference between different serum batch; Amplify circulation ratio poor; High to downstream purification processing requirement; Easily occur the vaccine quality hidden danger that serum goes out complicated ingredient and causes relating to Biosafety and public health problem.In addition, current global serum supply is becoming tight, and this have impact on the production efficiency of vaccine enterprise greatly.Therefore become the development trend of PK-15 cell cultures less with serum, the research and apply of low serum and serum-free cell culture technology has been put into 12 national science and technology supporting plan main projects simultaneously as far as possible.
Prior art usually through plant-derived albumen compositions such as animal source composition and soy bean protein hydrolysate such as interpolation bovine serum albumin, Transferrins,iron complexes, Regular Insulin, recombinant human epidermal growth factors, thus reduces the usage quantity of serum.But the security of above-mentioned protein to biological products (as vaccine) has a significant impact.
Summary of the invention
Invention broadly provides a kind of low serum protein-free medium being suitable for PK-15 Growth of Cells and preparation method thereof, have without albumen, non-animal derived composition, clearly several large advantage of Chemical Composition, scientific research can reduce the interference of serum to experimentation and result, use substratum of the present invention, make to be carry out under 2% low-serum-concentration condition in the culturing process of PK-15 cell, decrease the consumption of serum.Its technical scheme is as follows: a kind of low serum protein-free medium being suitable for PK-15 Growth of Cells, and it comprises following component:
Amino acid moiety comprises following component:
Vitamin moieties comprises following component:
Inorganic salt part comprises following component:
Trace element part comprises following component:
Also comprise other components following:
Preferably, a kind of component of low serum protein-free medium of the PK-15 of being suitable for Growth of Cells is as follows: amino acid moiety comprises following component:
Vitamin moieties comprises following component:
Inorganic salt part comprises following component:
Trace element part comprises following component:
Also comprise other components following:
Be suitable for a preparation method for the low serum protein-free medium of PK-15 Growth of Cells, comprise the steps:
(1) getting dried each component is respectively charged in ball grinder, and ball milling 1.5-3 hour, obtains culture medium dry powder;
(2) culture medium dry powder that step (1) is obtained is dissolved in ultrapure water, and carries out constant volume, with the sterilizing of bacteriological filtration membrane filtration, the low serum of PK-15 Growth of Cells must be suitable for without protein liquid substratum.
Preferably, in step (1), Ball-milling Time is 2 hours, and in step (2), the aperture of bacteriological filtration film is 0.22 μm.
Adopt above-mentioned substratum and preparation method thereof, the present invention has the following advantages:
(1) substratum of the present invention, have without albumen, non-animal derived composition, clearly several large advantage of Chemical Composition, scientific research can reduce the interference of serum to experimentation and result, industrial production can be enhanced productivity, ensure quality product, on the basis remaining traditional process equipment, production cost is reduced by the usage quantity reducing serum, the most important thing is simultaneously, it does not change the adherent growth state of PK-15 cell, substratum provided by the invention is applicable to the adherent culture of cell, with other combine with technique such as microcarriers, can in shaking flask, bio-reactors etc. carry out pilot scale culture, thus be widely used in suitability for industrialized production,
(2) the low serum protein-free medium of PK-15 cell 2% of the present invention and basic medium add 10% foetal calf serum and have carried out cell cultures and compare, easier decreasing pollution, be conducive to the separation of derived product, security is high, it is close that the growth curve of its cell cultures and virus connect malicious multiplication capacity, and without marked difference, substratum of the present invention does not change traditional processing technology, do not need the production unit more renewed, can be used for existing domestic production;
(3) substratum of the present invention overcomes during PK-15 cell routine is cultivated the shortcoming that must use high-content calf serum ability growth promoting effects, poor stability, the process of culture medium culturing PK-15 cell of the present invention is carried out under 2% low-serum-concentration, decreases the consumption of serum.
Accompanying drawing explanation
Fig. 1 is state graph under the PK-15 microcytoscope of DMEM high sugar+10% calf serum concentration cultivation;
Fig. 2 adds state graph under the bimestrial PK-15 microcytoscope of 2% calf serum cultured continuously for using low blood serum medium of the present invention;
Fig. 3 uses low blood serum medium of the present invention to add the growth curve of the PK-15 cell of 2% calf serum and the growth curve comparison diagram of the PK-15 cell using existing 10% calf serum DMEM to cultivate.
Embodiment
Embodiment 1
The present embodiment is for the preparation of being suitable for the low serum of PK-15 Growth of Cells without albuminous cell substratum.1. the formula of substratum
Table 1 culture medium prescription
2. preparation method
(1) getting dried each component in formula is respectively charged in ball grinder, and ball milling 2 hours, obtains culture medium dry powder;
(2) the culture medium dry powder 15.62g that step (1) is obtained is dissolved in 900ml ultrapure water, then 1L is settled to ultrapure water, with the bacteriological filtration membrane filtration sterilizing that aperture is 0.22 μm, the low serum that must be suitable for PK-15 Growth of Cells, without protein liquid substratum, refrigerates for subsequent use at 6 DEG C.
Embodiment 2
The present embodiment is for the preparation of being suitable for the low serum of PK-15 Growth of Cells without albuminous cell substratum.
1. the formula of substratum
Table 2 culture medium prescription
2. preparation method
(1) getting dried each component in formula is respectively charged in ball grinder, and ball milling 1.5 hours, obtains culture medium dry powder;
(2) culture medium dry powder that step (1) is obtained is dissolved in 900ml ultrapure water, then 1L is settled to ultrapure water, with the bacteriological filtration membrane filtration sterilizing that aperture is 0.22 μm, the low serum that must be suitable for PK-15 Growth of Cells, without protein liquid substratum, refrigerates for subsequent use at 8 DEG C.
Embodiment 3
The present embodiment is for the preparation of being suitable for the low serum of PK-15 Growth of Cells without albuminous cell substratum.
1. the formula of substratum
Table 3 culture medium prescription
2. preparation method
(1) getting dried each component in formula is respectively charged in ball grinder, and ball milling 3 hours, obtains culture medium dry powder;
(2) culture medium dry powder that step (1) is obtained is dissolved in 900ml ultrapure water, then 1L is settled to ultrapure water, with the bacteriological filtration membrane filtration sterilizing that aperture is 0.22 μm, the low serum that must be suitable for PK-15 Growth of Cells, without protein liquid substratum, refrigerates for subsequent use at 4 DEG C.
Embodiment 4
The present embodiment is for illustration of the laboratory using method of low serum without albuminous cell medium liquid being applicable to PK-15 Growth of Cells of the present invention.
1.PK-15 cell cultures
A) in existing DMEM high glucose medium (DMEM high glucose medium is known and the substratum containing each seed amino acid and glucose determined of composition), add the PK-15 cell that 10% concentration calf serum is cultivated, grow to and to be paved with bottom T25 square vase after about 80-90%, remove nutrient solution, use the aseptic PBS liquid (0.2ml/cm of not calcium-magnesium-containing
2) rinsing cell;
B) the aseptic Digestive system (0.2ml/cm containing 0.25% (w/v) trypsinase and 0.02% (w/v) EDTA is added
2) after, be placed in 5%CO
2, saturated humidity incubator, hatch for 37 DEG C and become circle, sucking-off Digestive system to cellular contraction, go down to posterity after adding substratum piping and druming evenly, inoculum density is 1 ~ 2 × 10
5cells/ml.
2. with substratum of the present invention, low serum domestication is carried out to PK-15 cell
Adopt direct method or progressively fall adaptation and the domestication that serum adjustment procedure carries out the low serum free culture system of PK-15 cell.
A) direct method: the cell that aforementioned stable grows directly is inoculated in low serum protein-free medium prepared by embodiment 1,2 or 3 and cultivates, substratum adds the calf serum of 2% when cell cultures, stablize and namely represent after energy continuous passage tame successfully until its growth conditions.
B) serum adjustment procedure progressively falling: is inoculated in by the cell that aforementioned stable grows in the substratum of the present invention containing 10% calf serum concentration and cultivates, progressively reduce the serum content in substratum, namely serum content is respectively from 10%, 5% is finally down to 2%, carry out the 2% low serum free culture system that cell continues, under each serum-concentration, cell carries out continuing to go down to posterity, cell is transferred in the substratum containing lower concentration serum after in stable condition, until adapt to low serum protein-free medium completely, the substratum of the present invention of above-mentioned use is embodiment 1, the substratum prepared in 2 or 3.
Interpretation of result: containing the PK-15 cell cultivated in 10% calf serum DMEM high glucose medium, cultivates in adopting direct method and progressively falling 2% low blood serum medium that PK-15 cell all can successfully adapt to from 10% calf serum cultivation the present invention by serum adjustment procedure.
Direct method domestication process time is short, but adaptive process incipient cell there will be obvious inadaptability, and does not occur too significantly incompatibility phenomenon progressively falling cell in serum adaptive process.No matter be direct or serum free culture system domestication falls in round-about way, the low serum protein-free medium in the present invention can well for PK-15 cell provides a suitable growing environment under 2% serum-concentration condition.
Fig. 1 is the PK-15 cell picture that DMEM high sugar+10% serum-concentration is cultivated, and Fig. 2 adds 2% calf serum cultured continuously bimestrial PK-15 cell picture for using low blood serum medium of the present invention.Contrasted from Fig. 1 and Fig. 2, adopt substratum of the present invention under 2% calf serum condition, be more suitable for the growth of PK-15 cell.
Embodiment 5
The present embodiment is more better than conventional use 10% calf serum concentration cultures culturing cell for illustration of the low serum of the PK-15 of being suitable for Growth of Cells of the present invention without albuminous cell culture medium culturing cell.
Cell growth curve measures: the DMEM high glucose medium cultured cells adding 10% calf serum by existing, add 2% calf serum embodiment 1 in the cell of culture medium culturing be inoculated in 12 orifice plates respectively, 1 × 10
5cells/ hole, and use corresponding culture medium culturing, survey a cell density every 24h, often kind of cell gets 3 holes at every turn, after trysinization, counts with Trypan Blue.
Interpretation of result: two kinds of culture system cultured cells growth power curves, as that shown in fig. 3, the PK-15 cellular form that the speed of growth of the PK-15 cell of 2% low serum free culture system and 10% calf serum DMEM high sugar is cultivated does not have notable difference, and the PK-15 cell of 2% low serum free culture system is compared to the PK-15 vitro growth rates of the high sugar cultivation of 10% calf serum DMEM and cell quantity also slightly advantage.It can thus be appreciated that the substratum adopting the present invention to prepare is applicable to 2% low serum free culture system environment, can reduce amount of serum, cost-saving.
To one skilled in the art, according to technical scheme described above and design, other various corresponding change and deformation can be made, and all these change and deformation all should belong within the protection domain of the claims in the present invention.
Claims (4)
1. be suitable for a low serum protein-free medium for PK-15 Growth of Cells, it is characterized in that: it comprises following component:
Amino acid moiety comprises following component:
Vitamin moieties comprises following component:
Inorganic salt part comprises following component:
Trace element part comprises following component:
Also comprise other components following:
Thymidine 0.01-1mg/L xanthoglobulin 0.01-1mg/L
Linolic acid 0.01-1mg/LD-glucose 3000-5000mg/L
Phenol red 1-20mg/LHEPES1000-4000mg/L.
2. a kind of low serum protein-free medium being suitable for PK-15 Growth of Cells according to claim 1, is characterized in that: it comprises following component:
Amino acid moiety comprises following component:
Vitamin moieties comprises following component:
Inorganic salt part comprises following component:
Trace element part comprises following component:
Also comprise other components following:
Thymidine 0.0635mg/L xanthoglobulin 0.042mg/L
Linolic acid 0.042mg/LD-glucose 4500mg/L
Phenol red 15mg/LHEPES2979mg/L.
3. be suitable for a preparation method for the low serum protein-free medium of PK-15 Growth of Cells as claimed in claim 1, it is characterized in that: comprise the steps:
(1) getting dried each component is respectively charged in ball grinder, and ball milling 1.5-3 hour, obtains culture medium dry powder;
(2) culture medium dry powder that step (1) is obtained is dissolved in ultrapure water, and carries out constant volume, with the sterilizing of bacteriological filtration membrane filtration, the low serum of PK-15 Growth of Cells must be suitable for without protein liquid substratum.
4. the preparation method being suitable for the low serum protein-free medium of PK-15 Growth of Cells according to claim 3, is characterized in that: in step (1), Ball-milling Time is 2 hours, and in step (2), the aperture of bacteriological filtration film is 0.22 μm.
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Cited By (4)
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| CN106011066A (en) * | 2016-07-07 | 2016-10-12 | 温州生物材料与工程研究所 | Method for large-scale preparation of human PC-3 cells |
| CN107217029A (en) * | 2017-06-08 | 2017-09-29 | 浙江美保龙生物技术有限公司 | A kind of cell culture fluids of PK 15 and preparation method thereof, application method |
| CN108103003A (en) * | 2017-12-12 | 2018-06-01 | 四川百诺吉科技有限公司 | A kind of serum free medium and preparation method thereof for adapting to the full suspension growths of PK-15 and cell suspend acclimation method entirely |
| CN111518768A (en) * | 2020-04-14 | 2020-08-11 | 四川百诺吉科技有限公司 | Low-serum culture medium suitable for LMH cell adherent culture and preparation method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106011066A (en) * | 2016-07-07 | 2016-10-12 | 温州生物材料与工程研究所 | Method for large-scale preparation of human PC-3 cells |
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| CN108103003A (en) * | 2017-12-12 | 2018-06-01 | 四川百诺吉科技有限公司 | A kind of serum free medium and preparation method thereof for adapting to the full suspension growths of PK-15 and cell suspend acclimation method entirely |
| CN111518768A (en) * | 2020-04-14 | 2020-08-11 | 四川百诺吉科技有限公司 | Low-serum culture medium suitable for LMH cell adherent culture and preparation method thereof |
| CN111518768B (en) * | 2020-04-14 | 2023-11-21 | 四川百诺吉科技有限公司 | Low-serum culture medium suitable for LMH cell wall-attached culture and preparation method thereof |
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Application publication date: 20151216 |