CN102409020A - Carrier for non-woven fabric/polyester fiber cultured cells and using method thereof - Google Patents
Carrier for non-woven fabric/polyester fiber cultured cells and using method thereof Download PDFInfo
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- CN102409020A CN102409020A CN201010292386XA CN201010292386A CN102409020A CN 102409020 A CN102409020 A CN 102409020A CN 201010292386X A CN201010292386X A CN 201010292386XA CN 201010292386 A CN201010292386 A CN 201010292386A CN 102409020 A CN102409020 A CN 102409020A
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Abstract
The invention relates to a carrier for culturing cells by non-woven fabrics/polyester fibers and a using method thereof. The method specifically comprises the following steps: 1) non-woven fabric/polyester fiber cell culture carrier; 2) a method for processing and manufacturing a non-woven fabric/polyester fiber cell culture carrier; 3) a process and a method for cell culture using a nonwoven fabric/polyester fiber cell culture carrier in a cell culture system. The invention uses a material which is low in price and suitable for adherent growth of cells to prepare a cell carrier and culture cells, thereby greatly reducing the cost of the cell carrier and the cell culture; carriers of various geometries prefabricated from this material can provide greater surface area for growing greater numbers of cells; meanwhile, according to different requirements of specific cell culture processes, the material can be processed into carriers with different shapes and sizes, and a larger space is provided for process adjustment and optimization.
Description
Technical field
The present invention relates to biological technical field, specifically is that a kind of non-woven fabrics/polyester fiber culturing cell is with carrier and use its technology of carrying out cell cultures and method.
Background technology
It is a kind of technology and method that can the large scale culturing attached cell that cell carrier is cultivated, and is the main method that current adherent dependent form cell large scale is cultivated.The mounting medium that does not have toxic side effect through pair cell; Cell is attached at carrier surface or inside, can improve cell cultures area and efficient greatly, such as the microcarrier culture technique; Have homogeneous phase and cultivate the advantage that has dull and stereotyped cultivation and suspension culture concurrently; Culture condition (temperature, pH value, gas concentration lwevel etc.) is control easily, and culturing process systematize, robotization, is difficult for contaminated.But present cell carrier (for example cytodex series microcarrier) on the market costs an arm and a leg mostly, is inappropriate for the large-scale industrialization culturing cell.
Retrieval through to the prior art document is found; People such as M.Pohl scheidt are at " Vaccine " Volume 26; Issue 12; 17March 2008; Among " the Development and optimisation of a procedure for the production of Parapoxvirus ovis by large-scale microcarrier cell culture in a non-animal; non-human and non-plant-derived medium " that delivers on the Pages 1552-1565 (" the extensive microcarrier cell cultures of non-animal/people/plant-sourced nutrient solution is produced the process development of Parapoxvirus ovis and optimized "); Reported that use Cytodex-
microcarrier has carried out 50L bovine kidney cells BK KL3A strain reactor drum suspension culture; And industrial large-scale engineering amplified done fundamental research, and this large-scale cell cultures and the activity in production of repeatability need that the microcarrier of labor, commercialization microcarrier commonly used at present have that cytodex series, biosilon are serial, solo microcarrier etc.; These microcarriers are concerning being used for cell cultures; Price is often too expensive, and it is big to be used for the scale operation cost, is not suitable for carrying out suitability for industrialized production; Simultaneously, as the moulding commodity, the shape of these cell carriers and size are fixing, are difficult for changing, and have influenced design, improvement and the optimization of corresponding technology.
Summary of the invention
In order to overcome the defective of above-mentioned existing common cell carrier; The contriver is through research and development; Provide and utilized that a kind of price is low, non-woven fabrics/polyester fiber of being easy to cutting processing is as cell culture vector; In cell culture system, implement the attached cell carrier and cultivate, help reducing the cost that large-scale industrialization attached cell carrier is cultivated.
The cell attachment growing carrier that the present invention uses is a kind of non-woven fabrics/polyester fiber.Non-woven fabrics (English name: Non Woven) claim tNonwovens again.Be to constitute by fiber orientation or at random; It is a kind of need not spinning cotton and weaving cloth and the fabric that forms; Just textile staple or long filament are carried out orientation or support row at random, form fibre net structure, adopt machinery then, heat is sticking or method such as chemistry is reinforced and formed.Because of the outward appearance with cloth is called cloth with some performance.Non-woven fabrics is environment-friendly materials of new generation, has protection against the tide, ventilative, pliable and tough, light weight, not combustion-supporting, easy decomposition, nontoxic nonirritant, rich color, cheap, capable of circulation characteristics such as use again.The filamentary material of making non-woven fabrics mainly contains Vestolen PP 7052 or trevira etc.Wherein, the polyester that formed by organic dibasic acid and divalent alcohol polycondensation of trevira is through the synthon of spinning gained.It is with pure terephthalic acid (PTA) or DMT. Dimethyl p-benzenedicarboxylate (DMT) and terepthaloyl moietie (EG) fiber-forming polymer--the polyethylene terephthalate (PET) that to be raw material make through esterification or transesterify and polycondensation, the fiber of processing through spinning and aftertreatment.
It is the different shape and big or small cell carrier that material becomes that the present invention promptly is to use non-woven fabrics/polyester fiber, the technology and the method for carrying out cell cultures preparation virus or producing vaccine.
The present invention includes following steps:
1) non-woven fabrics/polyester fiber material is suitably handled (comprise and carry out various surface treatments) with physics or chemical mode, to be more conducive to cell attachment or growth or to improve the strength of materials; Simultaneously, different shapes and size, various cell growing carrier that have certain intensity, that be used for cell cultures are manufactured in shearing.
2) in cell culture system (comprising various Tissue Culture Flasks, bag, bio-reactor etc.), as carrier,, carry out cell cultures with suitable cell culture fluid (base) with the processed goods (comprising section, fragment, particle etc.) of above-mentioned non-woven fabrics/polyester fiber.
3) obtain cell growth or metabolite data; Perhaps, harvested cell or cell produce thing.
The present invention is that material becomes cell culture vector with non-woven fabrics/polyester fiber, carries out cell cultures, has the following advantages:
1, has higher cell cultures specific surface area, thereby can increase the cell total amount in the cell culture system.For example, be applied to cell cultures and roll bottle inside, through increasing the single cell cultures area that rolls bottle, reduce and roll a bottle quantity, thereby reduce labour intensity, and reduce the cell infection probability.
2, the shearing force that cell bore is less, and can guarantee higher nutrient utilization and oxygen transport.For example in being applied to bed technology culturing cell technology, cell can firmly be attached at non-woven fabrics/polyester fiber material surface and inner, and nutrient solution can improve the utilization ratio of nutritive ingredient through between carrier, flowing; Simultaneously, cell is embedded in the carrier inside fiber, has reduced the damage of outside shearing force pair cell, thereby can improve the oxygen transfer efficiency through strengthening stir speed (S.S.) or the fluid speed of flowing through.
3, reduce cost: non-woven fabrics/polyester fiber is compared with the commercialization cell carrier that tradition is common as carrier, and is cheap, thereby can reduce the cell cultures cost, is more suitable for cultivating in the extensive attached cell carrier of industrialization.
4, cheap, can disposablely use, reduced the crossed contamination between production batch.
5, non-woven fabrics/polyester fiber material is easy to be cut into different shape and size, such as circle, trilateral, square, rhombus or the like, has increased the scope of using greatly, the space of having improved process optimization.
Description of drawings
Fig. 1 is a kind of shape--the disc of cell carrier of the present invention;
Fig. 2 is a kind of shape--the lengthy motion picture shape of cell carrier of the present invention;
Fig. 3 is a kind of shape of cell carrier of the present invention--folding rhombus;
Fig. 4 is a kind of shape--the debris chip shape of cell carrier of the present invention.
Embodiment
Specific embodiment 1
Adopt continous pouring fixed bed cultured method in present embodiment, in bio-reactor, cultivate amplification vero cell, inoculate, prepare rabies virus then, produce hydrophobia.Non-woven fabrics/polyester fiber is made into the circular shaped patches body that diameter is 6mm, as the cell growing carrier.
1, adopts (the Old Hickory of Reemay company of U.S. BBA Fiberweb; TN; USA) the non-woven fabrics polyester fiber of producing (Non Woven Polyester Fabric); Style no.2214 is as non-woven fabrics/polyester fiber material, through surface treatment and cut into the circular shaped patches body that diameter is 6mm.
2,1 vero cell of recovery (by the wide spectrum host cell that goes down to posterity that African green monkey kidney cell is cultivated, buy from Chinese pharmaceutical biological product and identify institute) from the seed cell storehouse went down to posterity, and increased with square vase in 48 hours.
What 3, the cell biological reactor drum was selected is to buy from the 5L bio-reactor Celligen of U.S. NBS company Plus, and working volume 3.5L adopts the basket stirring system of fixed bed.The diameter that in bio-reactor, adds above-mentioned processing well cutting is that the round polyester of 6mm is cut into slices as cell carrier, and adds phosphoric acid buffer, sterilizes 30 minutes for 121 degrees centigrade.After the sterilization, the emptying phosphoric acid buffer adds the vero cell growth medium, and the control of beginning cell biological reactor condition.
4, with square vase amplification vero cell well, as the seed cell of cell biological reactor drum, with 2-5 * 10
5The density of/mL is inoculated on the non-woven fabrics/trevira circular shaped patches carrier in the bio-reactor, carries out amplification cultivation with the cell proliferation nutrient solution.Wherein the usage quantity of trevira circular shaped patches carrier is every liter of tank volume 35g, and employed cell proliferation nutrient solution adds 10% NBCS for the DMEM-F12 substratum.Cell biological reactor drum culture condition is potential of hydrogen (pH) value 7.2,37 ℃ of temperature, 50% of oxyty (DO) air saturation, stirring velocity 120rpm.
5, when the vero cell grows to certain density, generally after inoculation 7-13 days, use cell instead and keep substratum, with 0.005-0.125MOI (virus numbers that is used to infect and the ratio of cell number) infection multiplicity inoculation rabies virus 7aG strain, make cell infection; The used cell maintenance culture solution of cell uses the DMEM-F12 substratum to add that 1% calf serum is formulated; The condition of setting bioreactor culture is a pH value 7.6,36 ℃ of temperature, and 50% of oxyty (DO) air saturation, stirring velocity is 100rpm.Continous pouring results 20 days, virus titer remains on more than the 81gLD50/mL.
6, the viral liquid of results was with beta-propiolactone deactivation in 1: 4000.
7, be that filler carries out chromatography purification with sepharose 4 fast flow, collect the antigen peak.
8, virus stock solution used adds 1% rHSA after the purifying degerming, sucrose is that protective material is processed liquid preparation.
9, titration of virus, efficacy determinations, determining the protein quantity, the bovine serum albumin determination of residual amount, residual DNA is measured according to " 2005 editions related requests of Chinese pharmacopoeia carry out.
Specific embodiment 2
Adopt the method for continous pouring suspension culture in present embodiment, express the proteic recombination Chinese hamster ovary celI of a kind of gene recombinant human of secretion outside the born of the same parents but in bio-reactor, cultivate, results contain target recombinant human protein's results liquid continuously.Non-woven fabrics/polyester fiber is made into the fines body, as the cell growing carrier.
1, nonwoven cloth material adopts the trevira non-woven fabrics that domestic certain company produces, and material through suitable broken cutting, is become fines shape carrier, as cell culture substrate.
What 2, bio-reactor was selected is Dutch Applikon autoclavable bioreactor 15L reactor drum, working volume 11L.Fines shape non-woven fabrics cell carrier is washed with PBS, and expansion is spent the night, and rinsing back several times adds bio-reactor, autoclaving.Add aseptic cell growth medium, soak carrier.
3,1 recombinaant CHO cell of recovery from produce cell bank is cultivated amplification with square vase.
4, with the good recombinaant CHO cell of square vase amplification amplification, with 3-6 * 10
5The density of/mL is inoculated in the bio-reactor jar that fines shape non-woven fabrics/polyester fiber carrier is housed; Carry out the carrier suspension culture with cell culture fluid; Wherein the usage quantity of non-woven fabrics/polyester fiber carrier is every liter of tank volume 45g, and employed cell culture fluid adds 10% NBCS for the DMEM-F12 substratum.Cell culture condition is pH value 7.0-7.2,37 ℃ of temperature, 50% of oxyty (DO) air saturation, stirring velocity 15-35rpm.
5, cultivated about 8 days the inoculation back, through the cell/carrier cut-off equipment of bio-reactor, and continuous drawing cell culture harvest liquid, the equivalent constant speed adds fresh cell culture fluid continuously simultaneously, comes into effect continous pouring and cultivates.
6, information such as the stream of cells vector after damming according to the cell motility rate in the reactor drum and cell density, cell/carrier, cellular metabolism data, product protein expression situation are regulated irrigation rate, implementation process optimization.
7, resulting results liquid is carried out ultrafiltration and concentration, remove a certain amount of moisture.
8, the results liquid after concentrating is carried out protein purification.
9, make protein formulation according to the drug technique rules.
More than disclosedly be merely several specific embodiment of the present invention, but the present invention is not limited thereto, any those skilled in the art can think variation, all should drop in protection scope of the present invention.
Claims (7)
- One kind with non-woven fabrics/polyester fiber culturing cell with carrier and method of use thereof, it is characterized in that, may further comprise the steps:1) non-woven fabrics/polyester fiber material is comprised the processing of various surface treatments with physics or chemical mode, to be more conducive to cell attachment or growth or to improve the strength of materials; Simultaneously, different shapes and size, various cell growing carrier that have certain intensity, that be used for cell cultures are manufactured in shearing;2) in the cell culture system that comprises various Tissue Culture Flasks, bag, bio-reactor, with above-mentioned polyester fiber material be processed into comprise section, fragment, particle processed goods as carrier, with cell culture fluid/base, carry out cell cultures;3) obtain cell growth or metabolite data; Perhaps, harvested cell or cell produce thing.
- 2. the method for claim 1; It is characterized in that; Said cell can be the one or more combinations in following: kidney cell, CEF, the insect cell (comprising by silkworm moth (anteraea eucalypti) gonad cell, Aedes albopictus (Aedes albopictus Skuse) cell Aa2-678, small cabbage moth (Plutell xylostella L innaeus) cell BCIRL-Px2-HNU3, greedy leaf moth (Spodoptera frugiperda) the gonad cell Sf21 in meadow and clone strain Sf9 and trichoplusia ni (Trichop lusiani) embryonic cell BTI-Tn5-BI-4 (the commercial High of being referred to as Five)) of Chinese hamster ovary celI (Chinese hamster ovary cell/Chinese hamster ovary cell), BHK21 cell (young golden hamster kidney cell/Baby hamster kidney cell), Marc145 cell (clone RhMK/Maacague kidney cell), Vero cell (African green monkey kidney cell/Kidney cells of Afrcan Green Monkey), F81 cell (feline kidney cells/Kidney cells of Cat, Felis catus), pig kidney continuous cell line (PK-15 or IBRS-2) or other responsive pig source cell, ST cell (tire pig testis cell/Testis cells of swine), bull testis cell (Testis cells of fetus boyine), cattle and sheep dog etc.; Above-mentioned various cell should comprise its each under all hypotype cell strains and various domestication thereof, genetic engineering modified type cell strain.
- 3. according to claim 1 or claim 2 method is characterized in that, comprising: said cell cultures comprises that animal tissues cultivates.
- 4. the method for claim 1; It is characterized in that; Cell culture system comprises that stirring type bioreactor, airlift bioreactor, cell suspension or wall-attaching type growth bio-reactor, intermittent type or stream add the biological reactor for cell culture of formula or continous pouring formula bio-reactor, wave bio-reactor, and comprises the various forms of various Tissue Culture Flasks, bag system, disposable plastic bag system and based on the cell culture system or the device of various principles.
- 5. method as claimed in claim 2 is characterized in that, comprising: the cell cultures mode comprises common adherent fixed-bed type cell cultures, the floated cell cultures of carrier, with and various development mode.
- 6. polyester fiber cell carrier that can be used for culturing cell; It is characterized in that; Comprise: the polyester fiber class material of making this carrier comprises that polypropylene-base, polyester, viscose glue class, acrylic fibres, polyamide-based and other fibers comprise the sanction body after the surface treatment processing through chemistry or physics, and said carrier comprises through shearing or processing having different shapes and different big or small, the various cell growing carrier certain intensity of tool, that can be used for cell cultures.
- 7. polyester fiber cell carrier as claimed in claim 6 is characterized in that: cutting mode and cutting profile that said carrier is made by non-woven fabrics/polyester fiber material can change arbitrarily with concrete application; Can but be not limited to and be made into various shapes that it is contemplated that and make such as comprising circle, square, trilateral, chip, debris and size arbitrarily, said carrier can but to be not limited to be that spherical, bar-shaped, strip, sheet are at interior any regular and irregular shape carrier.
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| CN201010292386XA CN102409020A (en) | 2010-09-26 | 2010-09-26 | Carrier for non-woven fabric/polyester fiber cultured cells and using method thereof |
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| CN201010292386XA CN102409020A (en) | 2010-09-26 | 2010-09-26 | Carrier for non-woven fabric/polyester fiber cultured cells and using method thereof |
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| CN103289897A (en) * | 2013-06-08 | 2013-09-11 | 上海日泰医药设备工程有限公司 | Cell culture bottle |
| CN105660422A (en) * | 2016-04-20 | 2016-06-15 | 遵义医学院 | Tissue culture method for bletillastriata (thunb.) reichb.f. seedlings |
| CN106754627A (en) * | 2017-02-27 | 2017-05-31 | 南京新诺丹生物技术有限公司 | A kind of carrier for cultured cells |
| US10920185B2 (en) | 2016-05-31 | 2021-02-16 | Corning Incorporated | Vessels and spinner flasks with reduced impeller wobble for culturing cells |
| WO2021129472A1 (en) * | 2019-12-25 | 2021-07-01 | Cesco Bioengineering Co., Ltd. | A carrier for cell biomass production and cell culture device comprising the same |
| CN113728085A (en) * | 2019-02-05 | 2021-11-30 | 康宁股份有限公司 | Woven cell culture substrate |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103289897A (en) * | 2013-06-08 | 2013-09-11 | 上海日泰医药设备工程有限公司 | Cell culture bottle |
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| US10920185B2 (en) | 2016-05-31 | 2021-02-16 | Corning Incorporated | Vessels and spinner flasks with reduced impeller wobble for culturing cells |
| CN106754627A (en) * | 2017-02-27 | 2017-05-31 | 南京新诺丹生物技术有限公司 | A kind of carrier for cultured cells |
| CN113728085A (en) * | 2019-02-05 | 2021-11-30 | 康宁股份有限公司 | Woven cell culture substrate |
| WO2021129472A1 (en) * | 2019-12-25 | 2021-07-01 | Cesco Bioengineering Co., Ltd. | A carrier for cell biomass production and cell culture device comprising the same |
| GB2606928A (en) * | 2019-12-25 | 2022-11-23 | Cesco Bioengineering Co Ltd | A carrier for cell biomass production and cell culture device comprising the same |
| GB2606928B (en) * | 2019-12-25 | 2023-11-01 | Cesco Bioengineering Co Ltd | A carrier for cell biomass production and cell culture device comprising the same |
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Application publication date: 20120411 |