ROCK Method for efficient long-term cryopreservation of intestine organoid by a combination of organoid state and optimal treatment timing of Rho-kinase inhibitor
KOREA INSTITUTE OF RADIOLOGICAL & MEDICAL SCIENCES;한국원자력의학원
发明人:
LEE, SEUNG BUM,이승범,PARK, SUN HOO,박선후,HAN, SUNG HOON,한성훈,SHIM, SE HWAN,심세환,KIM, MIN JUNG,김민정,JANG, WON SUK,장원석,LEE, SUN JU,이선주,MYUNG, JAE KYUNG,명재경,LEE, SEUNG SOOK,이승숙,JIN, YOUNG WOO,진영우,LEE, SEUNG BUMKR,PARK, SUN HOOKR,HAN, SUNG HOONKR,SHIM, SE HWANKR,KIM, MIN JUNGKR,JANG, WON SUKKR,LEE, SUN JUKR,MYUNG, JAE KYUNGKR,LEE, SEUNG SOOKKR,JIN, YOUNG WOOK
申请号：
KR1020160109307
公开号：
KR1020180023640A
申请日:
2016.08.26
申请国别(地区):
KR
年份:
2018
代理人:
摘要：
The present invention provides a method for efficient long-term cryopreservation of an intestine organoid through complex treatment of intestine organoid state and Rho-kinase (ROCK) inhibitor adding time. A method for long-term cryopreservation of an organoid of the present invention comprises the steps of: in vitro culturing the organoid； adding a culture medium having a ROCK (Rho-kinase) inhibitor included in the cultured intact undissociated organoid； and cryopreserving the culture medium-added organoid. The method according to the present invention has confirmed that survival rate is high when a cryopreserved in vitro cultured intestine organoid is thawed after cryopreserving the in vitro cultured intestine organoid in the form of an intact intestine organoid compared to a single cell. The method according to the present invention has confirmed that a method of directly adding the ROCK inhibitor to a freezing medium has a remarkably high survival recovery rate compared to a method of performing a pretreatment and a treatment process after thawing the cryopreserved in vitro cultured intestine organoid when cryopreserving the in vitro cultured intestine organoid for a long time of 3 months or more. Further, the method according to the present invention has observed that morphological characteristics of an in vivo intestine having a crypt-villi structure are maintained as a result of analyzing characteristics of the intestine organoid cultured after being thawed. Therefore, the method according to the present invention is considered that a high efficiency cryopreservation method applied to the present invention can be applied to and used in effective treatment of intestine diseases and damage by providing a large amount of cells essential to organoid-based therapy.본 발명은 장 오가로이드 상태 및 ROCK 저해제 첨가시기의 복합처리를 통한 장 오가로이드의 효율적인 장기냉동동결보존방법을 제공한다. 본 발명에 따르면 생체 외에서 배양된 장 오가로이드를 단일세포 보다 온전한 오가로이드 (intact intestine organoid) 형태로 냉동동결보존 후 해동시 생존율이 높음을 확인하였다. 3개월 이상의 장기냉동동결시에는 RO
