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1. WO2019056217 - METHOD FOR CRYOPRESERVING VERO CELLS WITH HIGH VIABILITY

Publication Number WO/2019/056217
Publication Date 28.03.2019
International Application No. PCT/CN2017/102485
International Filing Date 20.09.2017
IPC
A01N 1/02 2006.01
AHUMAN NECESSITIES
01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
1Preservation of bodies of humans or animals, or parts thereof
02Preservation of living parts
C12N 5/071 2010.01
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
5Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
07Animal cells or tissues
071Vertebrate cells or tissues, e.g. human cells or tissues
CPC
A01N 1/02
AHUMAN NECESSITIES
01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES, AS HERBICIDES
1Preservation of bodies of humans or animals, or parts thereof
02Preservation of living parts
Applicants
  • 苏州北开生化设备有限公司 SUZHOU BEIKAI INSTRUMENTS CO., LTD. [CN]/[CN]
Inventors
  • 马荣华 MA, Ronghua
Agents
  • 苏州市方略专利代理事务所(普通合伙) SUZHOU FANGLVE PATENT AGENT FIRM (ORDINARY PARTNERSHIP)
Priority Data
Publication Language Chinese (ZH)
Filing Language Chinese (ZH)
Designated States
Title
(EN) METHOD FOR CRYOPRESERVING VERO CELLS WITH HIGH VIABILITY
(FR) PROCÉDÉ DE CRYOCONSERVATION DE CELLULES VERO À VIABILITÉ ÉLEVÉE
(ZH) 一种高存活率的Vero细胞的冻存方法
Abstract
(EN)
Provided is a method for cryopreserving Vero cells with high viability, the method comprising the steps of: sucking up a culture medium with a pipette, adding PBS to wash the Vero cells twice or three times, adding 0.38% trypsin to a cell culture dish, digesting for 3 to 5 min at 37ºC, adding 2 to 3 ml of a fresh culture medium, and pipetting the bottom of the cell culture dish using the pipette 40 to 50 times; transferring the liquid to a cell centrifuge tube, centrifuging for 3 to 10 min under the condition of 1000-1500 g/min, removing the supernatant using the pipette, adding a cryopreservation liquid, vertically pipetting the cells 40 to 50 times using the pipette, adding the cells into a cell cryopreservation tube, tightening the cap of the cryopreservation tube, and sealing the cryopreservation tube with a sealing film; and preserving for 10 to 30 min at 2 to 7ºC, and for another 40 to 60 min at -15 to -20ºC, and then transferring same into an environment of ≤-80ºC for cryopreservation.
(FR)
L'invention concerne un procédé de cryoconservation de cellules Vero à viabilité élevée, comprenant les étapes consistant à : aspirer un milieu de culture avec une pipette, ajouter un tampon phosphate salin (PBS) pour laver les cellules Vero deux ou trois fois, ajouter 0,38 % de trypsine à une boîte de culture cellulaire, faire digérer pendant 3 à 5 min à 37 °C, ajouter de 2 à 3 ml d'un milieu de culture frais, et pipeter le fond de la boîte de culture cellulaire à l'aide de la pipette 40 à 50 fois; transférer le liquide vers un tube de centrifugation cellulaire, centrifuger pendant 3 à 10 min dans des conditions de 1000 à 1500 g/min, éliminer le surnageant à l'aide de la pipette, ajouter un liquide de cryoconservation, pipeter verticalement les cellules 40 à 50 fois à l'aide de la pipette, ajouter les cellules dans un tube de cryoconservation cellulaire, serrer le capuchon du tube de cryoconservation, et sceller le tube de cryoconservation avec un film d'étanchéité; et le conserver pendant 10 à 30 min entre 2 et 7 °C, et encore pendant 40 à 60 min entre -15 et -20 °C, puis le transférer dans un environnement de ≤ -80 °C pour la cryoconservation.
(ZH)
本发明提供了一种高存活率的Vero细胞的冻存方法,用移液枪将培养基吸尽,加入PBS洗涤Vero细胞2-3次,向细胞培养皿中加入0.38%的胰蛋白酶,于37℃条件下消化3-5min,加入2-3ml的新鲜的培养基,使用移液枪吹打细胞培养皿底部40-50次;将液体移至细胞离心管中,在1000-1500g/min条件下离心3-10min,用移液枪去除上清液,加入冻存液,使用移液枪上下吹打细胞40-50次,加入到细胞冻存管中,拧紧冻存管盖,用封口膜封住冻存管;在2-7℃保存10-30min,再于-15--20℃保存40-60min,然后移入到≤-80℃环境进行细胞冻存。
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