KR102619405B1 - 세포 배양 시트 및 신경줄기세포의 분리 배양 방법 - Google Patents
세포 배양 시트 및 신경줄기세포의 분리 배양 방법 Download PDFInfo
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- KR102619405B1 KR102619405B1 KR1020210172850A KR20210172850A KR102619405B1 KR 102619405 B1 KR102619405 B1 KR 102619405B1 KR 1020210172850 A KR1020210172850 A KR 1020210172850A KR 20210172850 A KR20210172850 A KR 20210172850A KR 102619405 B1 KR102619405 B1 KR 102619405B1
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- glu
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- leu
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Abstract
본 발명은 세포 배양 시트 및 신경줄기세포 배양 방법에 관한 것으로, 구체적으로는 신경조직, 콜라겐 및 피브린을 포함하는 하이드로겔을 포함하고, 상기 하이드로겔은 ROCK 억제제가 투여되는 안정화 단계를 거친 것인, 세포 배양 시트 및 이를 포함는 신경줄기세포 배양 방법을 제공하는 것이다.
Description
본 발명은 세포 배양 시트 및 신경줄기세포 배양 방법에 관한 것으로, 구체적으로는 신경조직, 콜라겐 및 피브린을 포함하는 하이드로겔을 포함하고, 상기 하이드로겔은 ROCK 억제제가 투여되는 안정화 단계를 거친 것인, 세포 배양 시트 및 이를 포함는 신경줄기세포 배양 방법을 제공하는 것이다.
현재 세포치료제로 가장 많이 이용되는 줄기세포는 비교적 쉽게 얻을 수 있는 지방, 골수, 제대혈 등에서 분리한 중간엽줄기세포이다. 하지만 중간엽줄기세포의 경우 특정 신경세포로의 분화 효율이 떨어져 치료적 효과가 제한적인라는 것이 한계로 지적된다.
신경줄기세포(neural stem cell)는 뉴런(neuron) 및 신경교세포(glial cell) 등으로 분화가 가능하다. 최근 설치류뿐 아니라 사람의 뇌에도 신경줄기세포의 존재가 확인되었으며 지속적으로 뇌세포의 항상성 유지 및 재생을 담당하는 것으로 밝혀졌다.
퇴행성 신경질환 또는 신경손상 치료를 위해서 다양한 기원의 신경줄기세포를 활용한 치료제 개발이 시도되고 있으나, 치료효과를 얻기 위한 충분한 신경줄기세포의 확보에 많은 어려움이 있었다.
기존의 연구는 물리적, 화학적 방법을 이용하여 뇌조직에서 신경줄기세포를 분리 배양하는 방법이었으나, 뇌 조직 질량에 비례하여 1회에 분리되는 세포의 수율에 한계가 있었다. 또한 물리적, 화학적 방법을 사용하여 반복적으로 세포분리를 할 경우에는 세포손상이 발생할 수 있는 문제가 있다.
본 발명은 상기한 실정을 고려하여 소량의 뇌조직을 활용하여 신경줄기세포를 분리 및 배양하는 방법을 제공할 수 있고, 이를 통하여 질량 대비 1회 획득할 수 있는 신경줄기세포의 수율을 극대화할 수 있는 분리 및 배양 방법을 제공할 수 있다.
또한 본 발명은 미분화 상태로 신경줄기세포를 유지 및 증폭 배양할 수 있는 방법을 제공할 수 있다.
또한, 본 발명이 해결하고자 하는 과제들은 이상에서 언급된 과제로 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 통상의 기술자에게 명확하게 이해될 수 있을 것이다.
상기와 같은 목적을 달성하기 위하여 본 발명의 일 실시예에 의하면, 신경조직, 콜라겐 및 피브린을 포함하는 하이드로겔을 포함하고, 상기 하이드로겔은 ROCK 억제제가 투여되는 안정화 단계를 거친 것인, 세포 배양 시트에 관한 것이다.
또한 본 발명에서, 상기 신경조직은 뇌조직인 것일 수 있다.
또한 본 발명에서, 상기 하이드로겔 100 중량부에 대하여, 상기 콜라겐은 2 중량부 이하로 포함되는 것일 수 있다.
또한 본 발명에서, 상기 안정화 단계는 120rpm 이하의 범위에서 교반하는 과정을 포함하는 것일 수 있다.
또한 본 발명에서, 상기 안정화 단계는 2일 이상 7일 이하의 기간 동안 이루어지는 것일 수 있다.
본 발명의 다른 실시예에 의하면, 본 발명에 따른 세포 배양 시트에 대하여, 3일 이상 30일 이하의 기간 동안 배양하는 제1 배양 단계를 포함하는, 신경줄기세포 배양 방법을 제공할 수 있다.
또한 본 발명에서, 시트 용해제를 투여하고, 신경세포를 분리하는 분리 단계; 및 분리된 상기 신경세포를 배양하는 제2 배양 단계;를 더 포함하는 것일 수 있다.
또한 본 발명에서, 상기 시트 용해제는 콜라게나제를 포함하고, 상기 시트 용해제 100 중량부에 대하여 콜라게나제를 0.01 중량부 이상 1 중량부 이하로 포함하는 것일 수 있다.
또한 본 발명에서, 상기 제2 배양 단계는 상기 신경세포의 밀집도가 60% 이상 90% 이하에서 설정된 기준치 이상일 때까지 배양하는 것일 수 있다.
또한 본 발명에서, 상기 신경세포를 동결보존하는 단계를 더 포함하는 것일 수 있다.
본 발명의 또 다른 실시예에 의하면, 신경조직으로부터 유래된 신경줄기세포를 제공하는 것일 수 있다.
또한 본 발명에서, 상기 신경줄기세포는 SOX2, nestin, PAX6, DCX 및 Olig2로 이루어진 군에서 선택된 하나 이상의 마커를 발현하는 것일 수 있다.
본 발명의 뇌조직 유래 신경줄기세포는 뇌에서 항상성 유지와 재생을 위해서 활성 되는 신경모세포 특이 표지자를 발현하며, 뉴런 및 성상교세포, 희소돌기세포 등의 뇌를 구성하는 세포로 분화할 수 있는 능력을 나타낸다. 또한 소량의 뇌조직으로 상대적으로 많은 세포를 확보할 수 있어 이를 활용하여 퇴행성신경질환 및 뇌손상 치료를 위한 세포치료제로 개발하는데 적용될 수 있다.
본 발명의 효과들은 이상에서 언급된 효과로 제한되지 않으며, 언급되지 않은 또 다른 효과들은 아래의 기재로부터 통상의 기술자에게 명확하게 이해될 수 있을 것이다.
도 1은 본 발명의 실시예에 있어서 각기 다른 부위 뇌조직에서 신경줄기세포가 증폭되는 것을 나타낸 이미지이다.
도 2는 본 발명의 실시예에 있어서 콜라겐겔에서 신경줄기세포를 분리하여 메트리겔이 코팅된 배양접시에서 배양된 신경줄기세포의 형태를 나타낸 이미지이다.
도 3은 본 발명의 실시예에 있어서 분리된 신경줄기세포가 신경모세포 특이 표지자를 발현하는지 확인하기 위하여 면역형광염색을 실시한 결과이다.
도 4는 본 발명의 실시예에 있어서 분리된 신경줄기세포가 뉴런 및 성상교세포, 혈관평활근세포, 대식세포 특이 표지자를 발현하는지 확인하기 위하여 면역형광염색을 실시한 결과이다.
도 5는 본 발명의 실시예에 있어서 분리된 신경줄기세포가 뉴런 및 성상교세포, 혈관평활근세포, 대식세포 특이 표지자를 발현하는지 확인하기 위하여 면역형광염색을 실시한 결과이다.
도 2는 본 발명의 실시예에 있어서 콜라겐겔에서 신경줄기세포를 분리하여 메트리겔이 코팅된 배양접시에서 배양된 신경줄기세포의 형태를 나타낸 이미지이다.
도 3은 본 발명의 실시예에 있어서 분리된 신경줄기세포가 신경모세포 특이 표지자를 발현하는지 확인하기 위하여 면역형광염색을 실시한 결과이다.
도 4는 본 발명의 실시예에 있어서 분리된 신경줄기세포가 뉴런 및 성상교세포, 혈관평활근세포, 대식세포 특이 표지자를 발현하는지 확인하기 위하여 면역형광염색을 실시한 결과이다.
도 5는 본 발명의 실시예에 있어서 분리된 신경줄기세포가 뉴런 및 성상교세포, 혈관평활근세포, 대식세포 특이 표지자를 발현하는지 확인하기 위하여 면역형광염색을 실시한 결과이다.
이하, 상기와 같은 목적을 달성하기 위하여 본 발명에 대하여 상세히 설명하기로 한다. 이에 앞서, 본 명세서 및 특허청구범위에 사용된 용어 또는 단어는 통상적이거나 사전적인 의미로 한정해서 해석되어서는 아니되며, 발명자는 그 자신의 발명을 가장 최선의 방법으로 설명하기 위해 용어의 개념을 적절하게 정의할 수 있다는 원칙에 입각하여 본 발명의 기술적 사상에 부합하는 의미와 개념으로 해석되어야만 한다. 따라서, 본 명세서에 기재된 실시예에 기재된 구성은 본 발명의 가장 바람직한 일 실시예에 불과할 뿐이고 본 발명의 기술적 사상을 모두 대변하는 것은 아니므로, 본 출원시점에 있어서 이들을 대체할 수 있는 다양한 균등물과 변형예들이 있을 수 있음을 이해하여야 한다.
한편, 본 명세서에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 명세서에서 개시된 다양한 요소들의 모든 조합이 본 출원의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.
본 발명의 일 구현 예에 따르면,
신경조직, 콜라겐 및 피브린을 포함하는 하이드로겔을 포함하고, 상기 하이드로겔은 ROCK 억제제가 투여되는 안정화 단계를 거친 것인, 세포 배양 시트에 관한 것이다.
본 발명에서, 상기 신경조직은 뇌조직인 것일 수 있다. 상기 신경조직은 뇌조직인 것일 수 있고, 상기 뇌조직은 피질(Cortex) 또는 측뇌실(lateral ventricle)에서 유래된 것일 수 있다.
본 발명에서, 상기 콜라겐은 콜라겐 1 내지 5에서 선택된 어느 하나 이상인 것일 수 있다.
본 발명에서, 상기 하이드로겔 100 중량부에 대하여, 상기 콜라겐은 2 중량부 이하로 포함되는 것일 수 있고, 상기 콜라겐은 1.5 중량부, 1.2 중량부, 1 중량부, 0.5 중량부, 0.3 중량부 또는 0.1 중량부 이하로 포함되는 것일 수 있다.
본 발명에서, 상기 피브린은 피브리노겐 및 트롬빈의 반응에 의해 생성된 것일 수 있다.
본 발명에서, 상기 ROCK 억제제는 ROCK 저해제, Rho 키나제 억제제 또는 Rho 관련 단백질 키나제 억제제라고도 하며, AT-13148, BA-210, beta-Elemene, Chroman 1, DJ4, Fasudil, GSK-576371, GSK429286A, H-1152, Hydroxyfasudil, Ibuprofen, LX-7101, Netarsudil, RKI-1447, Ripasudil, TCS-7001, Thiazovivin, Verosudil(AR-12286), Y-27632, Y-30141, Y-33075 및 Y-39983을 포함하는 군으로부터 선택된 어느 하나 이상인 것일 수 있으나, ROCK 경로를 억제하는 화합물이라면 제한없이 포함될 수 있다.
본 발명에서, 상기 안정화 단계는 120rpm 이하의 범위에서 교반하는 과정을 포함하는 것일 수 있고, 1rpm 이상 80rpm 이하의 범위, 60rpm 이하의 범위, 40rpm 이하의 범위, 30rpm 이하의 범위, 20rpm 이하의 범위, 10rpm 이하의 범위, 6rpm 이하의 범위 또는 1rpm 이하의 범위에서 교반하는 과정을 포함하는 것일 수 있다.
본 발명에서, 상기 안정화 단계는 2일 이상 7일 이하의 기간 동안 이루어지는 것이거나, 상기 안정화 단계는 2일 이상 3일 이하, 3일 이상 4일 이하, 4일 이상 5일 이하, 5일 이상 6일 이하 또는 6일 이상 7일 이하의 기간 동안 이루어지는 것일 수 있다.
본 발명의 다른 구현 예에 따르면,
본 발명에 따른 세포 배양 시트에 대하여, 3일 이상 30일 이하의 기간 동안 배양하는 제1 배양 단계를 포함하는, 신경줄기세포 배양 방법을 제공할 수 있다.
본 발명에서, 상기 제1 배양 단계는 3일 이상 5일 이하의 기간, 5일 이상 10일 이하의 기간, 10일 이상 15일 이하의 기간, 15일 이상 20일 이하의 기간, 20일 이상 25일 이하의 기간 또는 25일 이상 30일 이하의 기간 동안 배양하는 것일 수 있다.
본 발명에서, 시트 용해제를 투여하고, 신경세포를 분리하는 분리 단계; 및 분리된 상기 신경세포를 배양하는 제2 배양 단계;를 더 포함하는 것일 수 있다.
본 발명에서, 상기 시트 용해제는 콜라게나제를 포함하고, 상기 시트 용해제 100 중량부에 대하여 콜라게나제를 0.01 중량부 이상 1 중량부 이하로 포함하는 것일 수 있고, 상기 시트 용해제 100 중량부에 대하여 콜라게나제를 0.01 중량부 이상 0.02 중량부 미만, 0.02 중량부 이상 0.04 중량부 미만, 0.04 중량부 이상 0.06 중량부 미만, 0.06 중량부 이상 0.08 중량부 미만, 0.08 중량부 이상 0.1 중량부 미만, 0.1 중량부 이상 0.5 중량부 미만 또는 0.5 중량부 이상 1 중량부 이하로 포함하는 것일 수 있다.
본 발명에서, 상기 제2 배양 단계는 상기 신경세포의 밀집도가 60% 이상 90% 이하에서 설정된 기준치 이상일 때까지 배양하는 것일 수 있고, 상기 신경세포의 밀집도가 60% 이상 65% 미만의 기준치, 65% 이상 70% 미만의 기준치, 70% 이상 75% 미만의 기준치, 75% 이상 80% 미만의 기준치 또는 80% 이상 90% 이하의 기준치 이상일 때까지 배양하는 것일 수 있다.
본 발명에서, 상기 신경세포를 동결보존하는 단계를 더 포함하는 것일 수 있다.
본 발명의 일 예시에 따르면, 뇌조직을 재단하여 콜라겐겔에 함입하는 단계; 3일간 뇌조직의 세포 괴사를 방지하는 단계; 3주간 뇌조직에서 신경줄기세포를 증폭하는 단계; 콜라겐겔을 분해하여 증폭된 신경줄기세포를 분리하는 단계; 분리된 신경줄기세포를 배양접시에 파종하여 증폭하는 단계를 포함하는 신경모세포 표지자를 발현하는 신경줄기세포의 효율적인 분리 배양방법을 제공할 수 있다.
본 발명의 실시예에 있어서, 상기 뇌조직은 성인의 뇌조직일 수 있고, 재단한 크기는 1mm 이하 일 수 있다.
본 발명의 실시예에 있어서, 콜라겐겔의 농도는 0.05% 또는 0.1% 일 수 있으며, 다른 세포 외 기질 단백질을 첨가하여 겔을 제조시 총 비율은 0.2%(W/V)를 넘지 않는다.
본 발명의 실시예에 있어서, 뇌조직의 괴사를 방지를 위해 첨가하는 용액은 ROCK inhibitor(Y27632)에 국한되지 않는다.
본 발명의 실시예에 있어서, 콜라겐을 분해하는 효소 용액은 콜라게나제(collagenase) type 1, 2, 3, 4를 포함할 수 있다.
본 발명의 실시예에 있어서, 배양접시에 파종하여 시행되는 증폭배양은 matrigel, Poly-D-lysine, Poly-L-ornithine, fibronetin, laminin으로 코팅된 배양 접시를 사용할 수 있다.
본 발명의 실시예에 있어서, 분리된 신경줄기세포는 SOX2, nestin, PAX6, DCX 및 Olig2로 이루어진 군에서 하나 이상의 신경모세포 표지자에서 양성인 면역학적 특성을 나타낼 수 있다.
본 발명의 실시예에 있어서, 분리된 신경줄기세포는 Tuj-1(Neuron-specific class III beta-tubulin), GFAP(Glial fibrillary acidic protein)로 이루어진 군에서 음성인 면역학적 특성을 나타낼 수 있고, Olig2(Oligodendrocyte transcription factor 2) 로 이루어진 군에서 양성인 면역학적 특성을 나타낼 수 있다.
본 발명에서, 생략된 나머지 기재들은 본 발명의 나머지에서 기재된 바와 마찬가지로 해석될 수 있다.
본 발명의 또 다른 구현 예에 따르면,
본 발명의 신경조직으로부터 유래된 신경줄기세포를 제공하는 것일 수 있다.
본 발명에서, 하기 표 1에서와 같이, 상기 신경줄기세포는 SOX2, nestin, PAX6, DCX 및 Olig2로 이루어진 군에서 선택된 하나 이상의 마커를 발현하는 것일 수 있고, 상기 신경줄기세포는 SOX2 및 nestin 마커를 발현하는 것일 수 있고, 상기 신경줄기세포는 SOX2, nestin, PAX6, DCX 및 Olig2 마커를 모두 발현하는 것일 수 있다.
순번 | 마커 | 서열번호 |
1 | SOX2 | 서열번호 1 |
2 | nestin | 서열번호 2 |
3 | PAX6 | 서열번호 3 |
4 | DCX | 서열번호 4 |
5 | Olig2 | 서열번호 5 |
본 발명에서, 상기 SOX는 서열번호 1의 아미노산 서열을 포함하는 것일 수 있고, 배아줄기세포 및 신경줄기세포의 유지에 중요한 역할을 하여, 상기 마커를 발현하는 줄기세포의 경우 고품질의 정제된 신경줄기세포인 것을 확인할 수 있다.
또한, 상기 nestin은 서열번호 2의 아미노산 서열을 포함하는 것일 수 있고, 축삭의 방사상 성장과 관련된 신경세포에서 주로 발현되는 것으로, 상기 마커를 발현하는 줄기세포의 경우 고품질의 정제된 신경줄기세포인 것을 확인할 수 있다.
또한, 상기 PAX6은 서열번호 3의 아미노산 서열을 포함하는 것일 수 있고, 신경 및 표피 조직과 같은 외배엽 조직에서 유래하는 상동 구조의 발달을 위한 마스터 컨트롤 역할 하는 것으로, 상기 마커를 발현하는 줄기세포의 경우 고품질의 정제된 신경줄기세포인 것을 확인할 수 있다.
또한, 상기 DCX는 서열번호 4의 아미노산 서열을 포함하는 것일 수 있고, 신경전구세포 및 미성숙 뉴런에 의해 발현되는 미세소관 관련 단백질로, 상기 마커를 발현하는 줄기세포의 경우 고품질의 정제된 신경줄기세포인 것을 확인할 수 있다.
또한, 상기 Olig2는 서열번호 5의 아미노산 서열을 포함하는 것일 수 있고, 뇌와 척수의 제한된 영역에서 발현되는 것으로, 상기 마커가 발현되는 경우 고품질의 정제된 신경줄기세포인 것을 확인할 수 있다.
본 발명에서, 생략된 나머지 기재들은 본 발명의 나머지에서 기재된 바와 마찬가지로 해석될 수 있다.
이하, 본 발명의 실시예를 통해 본 발명을 더욱 상술하나 하기 실시예에 의해 본 발명이 제한되지 아니함은 자명하다.
1. 실시예
1.1. 뇌조직 추출
University of Wisconsin solutions(UW 용액) 또는 histidine-tryptophan-ketoglutarate(HTK 용액)의 장기보관용액에 보관된 뇌조직을 배양 접시로 옮긴 다음 100μg/mL의 겐타마이신(gentamycine)이 포함된 차가운 DPBS(Dulbecco's phosphate-buffered saline)를 사용하여 뇌조직을 3회 세척하였다. 다음으로 멸균된 블레이드를 사용하여 뇌 조직에 포함된 혈관을 박리하고, 혈관이 박리된 뇌 조직을 1mm 크기로 재단하여 50mL 튜브에 형체가 유지된 채로 옮겼다. 다음으로 DPBS를 사용하여 3회 세척한 후 아이스에 5분 간 보관하였다.
1.2. 콜라겐 하이드로겔의 준비
50mL 튜브에 10mL 용량의 0.1%의 콜라겐 용액을 제조하여 아이스에서 보관 후, 0.1%의 피브리노겐과 혼합하여 최종 0.2%의 혼합된 용액을 준비하였다. 다음으로 뇌조직이 포함된 튜브에 DPBS를 제거한 후, 0.2%의 콜라겐/피브리노겐 용액 6mL을 첨가한 다음, 트롬빈 0.5단위를 첨가하여 충분히 혼합하여 100mm 배양접시에 고르게 펼쳤다. 다음으로 상기 배양접시를 세포 배양 인큐베이터에 넣고 30분간 반응시켜 하이드로겔을 준비하였다.
1.3. 뇌조직 배양
상기 하이드로겔에 포함된 뇌조직을 배양하기 위해, 기본적으로 DMEM/F12와 신경세포배양용 배지(Neurobasal media)를 1:1 혼합한 배양 배지에 1% N2, 2% B27 without vitamin A, 1% Glutamax, 20μg/mL human epidermal growth factor(human EGF), 20μg/mL human basic fibroblast growth factor(human bFGF)를 첨가한 일반 배양 배지를 제조하였다. 뇌조직 배양 0일째 배양 상기 일반 배양 배지에 20μM ROCK inhibitor(Y27632)가 첨가된 배지를 사용하였고, 1시간 마다 2회 교환하였다. 다음 3일 동안 1일 1회 20μM ROCK inhibitor(Y27632)가 첨가된 배지로 교환하여 신경줄기세포의 생존을 강화하는 방법을 사용하였다. 상기 배지는 인큐베이터에서 60rpm으로 교반되었고, 상기 교반에는 회전식 쉐이커가 이용되었다.
1.4. 뇌조직 하이드로겔에서 신경줄기세포의 증폭배양
상기 배양된 뇌조직을 2주간 배양하였고, 이때 배지는 주 2회 교환하였다. 다음으로 도 1에 나타난 것처럼 신경줄기세포를 뇌조직에서 콜라겐 하이드로겔로 이동시켜 증폭배양시켰다. 그 결과 도 1에 나타난 것처럼, 피질(Cortex) 및 측뇌실(lateral ventricle)의 각기 다른 부위에서 4주에 걸쳐 신경줄기세포가 증식하는 것을 확인할 수 있었다.
1.5. 신경줄기세포의 회수
증폭된 신경줄기세포에서 잔존 배지를 제거한 후, DPBS를 사용하여 10분씩 3회 쉐이커에 올려 세척과정을 거쳤다. 다음으로 세척된 신경줄기세포에, 37℃워터배스로 데워진 0.05% 콜라게나제 타입 1(collagenase type 1)이 포함된 DMEM/F12 배지 10mL을 첨가하고, 다시 쉐이커에 올린 후 15분간 반응시켰다. 여기에 0.05% 콜라게나제가 포함된 DMEM/F12 배지를 5mL 추가로 첨가하고 5분 간 반응시켰고, 10mL 파이펫으로 콜라겐 하이드로겔을 충분히 녹인 후, 50mL 튜브에 옮겼다. 이후 옮겨진 튜브에 0.1%의 FBS를 첨가하여 효소반응을 정지시킨 후, 400 x g에서 원심분리 시켰다. 원심분리로 생성된 상층액을 제거하고, 조직 및 펠렛을 10μM의 Y27632가 첨가된 배양 배지에 현탁시켰다.
1.6. 메트리겔에서 신경줄기세포의 증폭배양
뇌조직 하이드로겔에서 신경줄기세포를 분리하여, 도 2에서와 같이 메트리겔(matrigel)이 코팅된 배양접시에서 배양하였다. 세포 배양을 준비하기 위하여, 클린벤치 내에서 25mL의 차가운 DMEM/F12 배지를 50mL 튜브에 옮긴 후, 50 ~ 125μL의 메트리겔을 첨가하여 0.2% ~ 0.5%로 정량하고 충분히 혼합한 다음, 100mm 배양접시에 6mL씩 넣고 고르게 펼친 후, 상온에서 1시간 유지하여 배양접시를 코팅하였다. 세포를 바이알(vial) 파종하기 직전 배양접시에 남은 메트리겔을 제거하고, 튜브에 담긴 세포를 10mL씩 배양접시에 파종하여 배양을 시작하였다. 배양 다음날, 부착되지 않은 조직과 콜라겐 조각을 DPBS로 세척하여 제거한 다음, 기본 배양 배지를 첨가하고 3 ~ 4일 마다 교체하여 증폭배양 시켰다.
1.6. 계대배양 및 동결보존
계대배양 및 동결보존은 80% 이상의 밀집도 및 배양접시 기준 1:4 ~ 1:6의 비율로 실시하였다. 먼저, 배양된 세포가 80% 이상의 밀집도를 보일 때, 세포를 37℃항온수조에서 데워진 DPBS를 사용하여 3회 세척시킨 후, 0.05% trypsin/EDTA가 첨가된 DPBS를 사용하여 세포를 바닥에서 탈락시켰다. 다음으로 1mL의 트립신 억제제(trypsin inhibitor)가 포함된 50mL 튜브를 준비하여, 탈락된 세포를 파이펫으로 옮긴 후, 300 x g에서 원심분리 하여 계대배양 및 동결보존을 위한 준비를 하였다.
계대배양의 경우, 상층액을 제거하고 남은 펠렛을 10μM Y27632가 포함된 배양 배지와 함께 100mm 배양접시에 나누어 실시하였다. 동결보존의 경우, 일반적으로 시중에 판매되는 줄기세포 전용 동결보존용액을 첨가하여 현탁한 후, 배양접시 기준으로 2vial로 나누어 실시하였다.
2. 평가예
2.1. 신경모세포 마커 확인
분리된 신경줄기세포가 신경모세포 특이 표지자(마커)를 발현하는지 확인하기 위하여 면역형광염색을 실시하였다. 그 결과 도 3에 나타난 것처럼, 분리된 신경줄기세포는 SOX2, nestin, PAX6, DCX 및 Olig2로 이루어진 군에서 선택된 하나 이상의 신경모세포 특이 표지자에서 양성인 면역학적 특성을 나타낼 수 있다.
2.2. 뉴런 및 성상교세포 마커 확인
분리된 신경줄기세포가 뉴런 및 성상교세포 특이 표지자를 발현하는지 확인하기 위하여 면역형광염색을 실시하였다. 그 결과 도 4에서 나타난 것과 같이, 분리된 신경줄기세포는 Tuj-1(Neuron-specific class III beta-tubulin), GFAP(Glial fibrillary acidic protein)로 이루어진 군에서 음성인 면역학적 특성을 나타낼 수 있고, Olig2(Oligodendrocyte transcription factor 2) 로 이루어진 군에서 양성인 면역학적 특성을 나타낼 수 있다.
2.3. 대식세포, 혈관평활근세포 및 혈관평활근세포 마커 확인
분리된 신경줄기세포가 뉴런 및 성상교세포, 혈관평활근세포, 대식세포 특이 표지자가 포함되었는지 확인하기 위하여 면역형광염색을 실시하였다. 그 결과 도 5에 나타난 것처럼, 분리된 신경줄기세포는 혈관내피세포 및 혈관평활근세포 특이 표지자인 SMA(smooth muscle actin) 마커가 음성이었고, 또한 대식세포(macrophage) 특이 표지자인 IBA-1(ionized calcium-binding adapter molecule 1) 마커가 음성인 것을 확인하여, 면역학적 특성을 확인할 수 있었다.
또한 분리된 신경줄기세포는 혈관내피세포 및 혈관평활근세포 특이 표지자인 CD31, CD34 마커가 음성이었고, 또한 대식세포(macrophage) 특이 표지자인 CD14 마커가 음성이었고, 또한 조혈계 세포 특이 표지자인 CD45 마커가 음성인 것을 확인하여, 면역학적 특성을 확인할 수 있었다.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 예를 들어, 단일형으로 설명되어 있는 각 구성 요소는 분산되어 실시될 수 있으며, 마찬가지로 분산된 것으로 설명되어 있는 구성 요소들도 결합된 형태로 실시될 수 있다.
본 발명의 범위는 청구범위에 의하여 나타내어지며, 청구범위 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.
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770 775 780
Leu Glu Pro Leu Lys Ser Leu Asp Gln Glu Ile Ala Arg Pro Leu Glu
785 790 795 800
Asn Glu Asn Gln Glu Phe Leu Lys Ser Leu Lys Glu Glu Ser Val Glu
805 810 815
Ala Val Lys Ser Leu Glu Thr Glu Ile Leu Glu Ser Leu Lys Ser Ala
820 825 830
Gly Gln Glu Asn Leu Glu Thr Leu Lys Ser Pro Glu Thr Gln Ala Pro
835 840 845
Leu Trp Thr Pro Glu Glu Ile Asn Gln Gly Ala Met Asn Pro Leu Glu
850 855 860
Lys Glu Ile Gln Glu Pro Leu Glu Ser Val Glu Val Asn Gln Glu Thr
865 870 875 880
Phe Arg Leu Leu Glu Glu Glu Asn Gln Glu Ser Leu Arg Ser Leu Gly
885 890 895
Ala Trp Asn Leu Glu Asn Leu Arg Ser Pro Glu Glu Val Asp Lys Glu
900 905 910
Ser Gln Arg Asn Leu Glu Glu Glu Glu Asn Leu Gly Lys Gly Glu Tyr
915 920 925
Gln Glu Ser Leu Arg Ser Leu Glu Glu Glu Gly Gln Glu Leu Pro Gln
930 935 940
Ser Ala Asp Val Gln Arg Trp Glu Asp Thr Val Glu Lys Asp Gln Glu
945 950 955 960
Leu Ala Gln Glu Ser Pro Pro Gly Met Ala Gly Val Glu Asn Glu Asp
965 970 975
Glu Ala Glu Leu Asn Leu Arg Glu Gln Asp Gly Phe Thr Gly Lys Glu
980 985 990
Glu Val Val Glu Gln Gly Glu Leu Asn Ala Thr Glu Glu Val Trp Ile
995 1000 1005
Pro Gly Glu Gly His Pro Glu Ser Pro Glu Pro Lys Glu Gln Arg Gly
1010 1015 1020
Leu Val Glu Gly Ala Ser Val Lys Gly Gly Ala Glu Gly Leu Gln Asp
1025 1030 1035 1040
Pro Glu Gly Gln Ser Gln Gln Val Gly Ala Pro Gly Leu Gln Ala Pro
1045 1050 1055
Gln Gly Leu Pro Glu Ala Ile Glu Pro Leu Val Glu Asp Asp Val Ala
1060 1065 1070
Pro Gly Gly Asp Gln Ala Ser Pro Glu Val Met Leu Gly Ser Glu Pro
1075 1080 1085
Ala Met Gly Glu Ser Ala Ala Gly Ala Glu Pro Gly Pro Gly Gln Gly
1090 1095 1100
Val Gly Gly Leu Gly Asp Pro Gly His Leu Thr Arg Glu Glu Val Met
1105 1110 1115 1120
Glu Pro Pro Leu Glu Glu Glu Ser Leu Glu Ala Lys Arg Val Gln Gly
1125 1130 1135
Leu Glu Gly Pro Arg Lys Asp Leu Glu Glu Ala Gly Gly Leu Gly Thr
1140 1145 1150
Glu Phe Ser Glu Leu Pro Gly Lys Ser Arg Asp Pro Trp Glu Pro Pro
1155 1160 1165
Arg Glu Gly Arg Glu Glu Ser Glu Ala Glu Ala Pro Arg Gly Ala Glu
1170 1175 1180
Glu Ala Phe Pro Ala Glu Thr Leu Gly His Thr Gly Ser Asp Ala Pro
1185 1190 1195 1200
Ser Pro Trp Pro Leu Gly Ser Glu Glu Ala Glu Glu Asp Val Pro Pro
1205 1210 1215
Val Leu Val Ser Pro Ser Pro Thr Tyr Thr Pro Ile Leu Glu Asp Ala
1220 1225 1230
Pro Gly Pro Gln Pro Gln Ala Glu Gly Ser Gln Glu Ala Ser Trp Gly
1235 1240 1245
Val Gln Gly Arg Ala Glu Ala Leu Gly Lys Val Glu Ser Glu Gln Glu
1250 1255 1260
Glu Leu Gly Ser Gly Glu Ile Pro Glu Gly Pro Gln Glu Glu Gly Glu
1265 1270 1275 1280
Glu Ser Arg Glu Glu Ser Glu Glu Asp Glu Leu Gly Glu Thr Leu Pro
1285 1290 1295
Asp Ser Thr Pro Leu Gly Phe Tyr Leu Arg Ser Pro Thr Ser Pro Arg
1300 1305 1310
Trp Asp Pro Thr Gly Glu Gln Arg Pro Pro Pro Gln Gly Glu Thr Gly
1315 1320 1325
Lys Glu Gly Trp Asp Pro Ala Val Leu Ala Ser Glu Gly Leu Glu Ala
1330 1335 1340
Pro Pro Ser Glu Lys Glu Glu Gly Glu Glu Gly Glu Glu Glu Cys Gly
1345 1350 1355 1360
Arg Asp Ser Asp Leu Ser Glu Glu Phe Glu Asp Leu Gly Thr Glu Ala
1365 1370 1375
Pro Phe Leu Pro Gly Val Pro Gly Glu Val Ala Glu Pro Leu Gly Gln
1380 1385 1390
Val Pro Gln Leu Leu Leu Asp Pro Ala Ala Trp Asp Arg Asp Gly Glu
1395 1400 1405
Ser Asp Gly Phe Ala Asp Glu Glu Glu Ser Gly Glu Glu Gly Glu Glu
1410 1415 1420
Asp Gln Glu Glu Gly Arg Glu Pro Gly Ala Gly Arg Trp Gly Pro Gly
1425 1430 1435 1440
Ser Ser Val Gly Ser Leu Gln Ala Leu Ser Ser Ser Gln Arg Gly Glu
1445 1450 1455
Phe Leu Glu Ser Asp Ser Val Ser Val Ser Val Pro Trp Asp Asp Ser
1460 1465 1470
Leu Arg Gly Ala Val Ala Gly Ala Pro Lys Thr Ala Leu Glu Thr Glu
1475 1480 1485
Ser Gln Asp Ser Ala Glu Pro Ser Gly Ser Glu Glu Glu Ser Asp Pro
1490 1495 1500
Val Ser Leu Glu Arg Glu Asp Lys Val Pro Gly Pro Leu Glu Ile Pro
1505 1510 1515 1520
Ser Gly Met Glu Asp Ala Gly Pro Gly Ala Asp Ile Ile Gly Val Asn
1525 1530 1535
Gly Gln Gly Pro Asn Leu Glu Gly Lys Ser Gln His Val Asn Gly Gly
1540 1545 1550
Val Met Asn Gly Leu Glu Gln Ser Glu Glu Val Gly Gln Gly Met Pro
1555 1560 1565
Leu Val Ser Glu Gly Asp Arg Gly Ser Pro Phe Gln Glu Glu Glu Gly
1570 1575 1580
Ser Ala Leu Lys Thr Ser Trp Ala Gly Ala Pro Val His Leu Gly Gln
1585 1590 1595 1600
Gly Gln Phe Leu Lys Phe Thr Gln Arg Glu Gly Asp Arg Glu Ser Trp
1605 1610 1615
Ser Ser Gly Glu Asp
1620
<210> 3
<211> 422
<212> PRT
<213> Homo sapiens
<400> 3
Met Gln Asn Ser His Ser Gly Val Asn Gln Leu Gly Gly Val Phe Val
1 5 10 15
Asn Gly Arg Pro Leu Pro Asp Ser Thr Arg Gln Lys Ile Val Glu Leu
20 25 30
Ala His Ser Gly Ala Arg Pro Cys Asp Ile Ser Arg Ile Leu Gln Val
35 40 45
Ser Asn Gly Cys Val Ser Lys Ile Leu Gly Arg Tyr Tyr Glu Thr Gly
50 55 60
Ser Ile Arg Pro Arg Ala Ile Gly Gly Ser Lys Pro Arg Val Ala Thr
65 70 75 80
Pro Glu Val Val Ser Lys Ile Ala Gln Tyr Lys Arg Glu Cys Pro Ser
85 90 95
Ile Phe Ala Trp Glu Ile Arg Asp Arg Leu Leu Ser Glu Gly Val Cys
100 105 110
Thr Asn Asp Asn Ile Pro Ser Val Ser Ser Ile Asn Arg Val Leu Arg
115 120 125
Asn Leu Ala Ser Glu Lys Gln Gln Met Gly Ala Asp Gly Met Tyr Asp
130 135 140
Lys Leu Arg Met Leu Asn Gly Gln Thr Gly Ser Trp Gly Thr Arg Pro
145 150 155 160
Gly Trp Tyr Pro Gly Thr Ser Val Pro Gly Gln Pro Thr Gln Asp Gly
165 170 175
Cys Gln Gln Gln Glu Gly Gly Gly Glu Asn Thr Asn Ser Ile Ser Ser
180 185 190
Asn Gly Glu Asp Ser Asp Glu Ala Gln Met Arg Leu Gln Leu Lys Arg
195 200 205
Lys Leu Gln Arg Asn Arg Thr Ser Phe Thr Gln Glu Gln Ile Glu Ala
210 215 220
Leu Glu Lys Glu Phe Glu Arg Thr His Tyr Pro Asp Val Phe Ala Arg
225 230 235 240
Glu Arg Leu Ala Ala Lys Ile Asp Leu Pro Glu Ala Arg Ile Gln Val
245 250 255
Trp Phe Ser Asn Arg Arg Ala Lys Trp Arg Arg Glu Glu Lys Leu Arg
260 265 270
Asn Gln Arg Arg Gln Ala Ser Asn Thr Pro Ser His Ile Pro Ile Ser
275 280 285
Ser Ser Phe Ser Thr Ser Val Tyr Gln Pro Ile Pro Gln Pro Thr Thr
290 295 300
Pro Val Ser Ser Phe Thr Ser Gly Ser Met Leu Gly Arg Thr Asp Thr
305 310 315 320
Ala Leu Thr Asn Thr Tyr Ser Ala Leu Pro Pro Met Pro Ser Phe Thr
325 330 335
Met Ala Asn Asn Leu Pro Met Gln Pro Pro Val Pro Ser Gln Thr Ser
340 345 350
Ser Tyr Ser Cys Met Leu Pro Thr Ser Pro Ser Val Asn Gly Arg Ser
355 360 365
Tyr Asp Thr Tyr Thr Pro Pro His Met Gln Thr His Met Asn Ser Gln
370 375 380
Pro Met Gly Thr Ser Gly Thr Thr Ser Thr Gly Leu Ile Ser Pro Gly
385 390 395 400
Val Ser Val Pro Val Gln Val Pro Gly Ser Glu Pro Asp Met Ser Gln
405 410 415
Tyr Trp Pro Arg Leu Gln
420
<210> 4
<211> 365
<212> PRT
<213> Homo sapiens
<400> 4
Met Glu Leu Asp Phe Gly His Phe Asp Glu Arg Asp Lys Thr Ser Arg
1 5 10 15
Asn Met Arg Gly Ser Arg Met Asn Gly Leu Pro Ser Pro Thr His Ser
20 25 30
Ala His Cys Ser Phe Tyr Arg Thr Arg Thr Leu Gln Ala Leu Ser Asn
35 40 45
Glu Lys Lys Ala Lys Lys Val Arg Phe Tyr Arg Asn Gly Asp Arg Tyr
50 55 60
Phe Lys Gly Ile Val Tyr Ala Val Ser Ser Asp Arg Phe Arg Ser Phe
65 70 75 80
Asp Ala Leu Leu Ala Asp Leu Thr Arg Ser Leu Ser Asp Asn Ile Asn
85 90 95
Leu Pro Gln Gly Val Arg Tyr Ile Tyr Thr Ile Asp Gly Ser Arg Lys
100 105 110
Ile Gly Ser Met Asp Glu Leu Glu Glu Gly Glu Ser Tyr Val Cys Ser
115 120 125
Ser Asp Asn Phe Phe Lys Lys Val Glu Tyr Thr Lys Asn Val Asn Pro
130 135 140
Asn Trp Ser Val Asn Val Lys Thr Ser Ala Asn Met Lys Ala Pro Gln
145 150 155 160
Ser Leu Ala Ser Ser Asn Ser Ala Gln Ala Arg Glu Asn Lys Asp Phe
165 170 175
Val Arg Pro Lys Leu Val Thr Ile Ile Arg Ser Gly Val Lys Pro Arg
180 185 190
Lys Ala Val Arg Val Leu Leu Asn Lys Lys Thr Ala His Ser Phe Glu
195 200 205
Gln Val Leu Thr Asp Ile Thr Glu Ala Ile Lys Leu Glu Thr Gly Val
210 215 220
Val Lys Lys Leu Tyr Thr Leu Asp Gly Lys Gln Val Thr Cys Leu His
225 230 235 240
Asp Phe Phe Gly Asp Asp Asp Val Phe Ile Ala Cys Gly Pro Glu Lys
245 250 255
Phe Arg Tyr Ala Gln Asp Asp Phe Ser Leu Asp Glu Asn Glu Cys Arg
260 265 270
Val Met Lys Gly Asn Pro Ser Ala Thr Ala Gly Pro Lys Ala Ser Pro
275 280 285
Thr Pro Gln Lys Thr Ser Ala Lys Ser Pro Gly Pro Met Arg Arg Ser
290 295 300
Lys Ser Pro Ala Asp Ser Gly Asn Asp Gln Asp Ala Asn Gly Thr Ser
305 310 315 320
Ser Ser Gln Leu Ser Thr Pro Lys Ser Lys Gln Ser Pro Ile Ser Thr
325 330 335
Pro Thr Ser Pro Gly Ser Leu Arg Lys His Lys Asp Leu Tyr Leu Pro
340 345 350
Leu Ser Leu Asp Asp Ser Asp Ser Leu Gly Asp Ser Met
355 360 365
<210> 5
<211> 323
<212> PRT
<213> Homo sapiens
<400> 5
Met Asp Ser Asp Ala Ser Leu Val Ser Ser Arg Pro Ser Ser Pro Glu
1 5 10 15
Pro Asp Asp Leu Phe Leu Pro Ala Arg Ser Lys Gly Ser Ser Gly Ser
20 25 30
Ala Phe Thr Gly Gly Thr Val Ser Ser Ser Thr Pro Ser Asp Cys Pro
35 40 45
Pro Glu Leu Ser Ala Glu Leu Arg Gly Ala Met Gly Ser Ala Gly Ala
50 55 60
His Pro Gly Asp Lys Leu Gly Gly Ser Gly Phe Lys Ser Ser Ser Ser
65 70 75 80
Ser Thr Ser Ser Ser Thr Ser Ser Ala Ala Ala Ser Ser Thr Lys Lys
85 90 95
Asp Lys Lys Gln Met Thr Glu Pro Glu Leu Gln Gln Leu Arg Leu Lys
100 105 110
Ile Asn Ser Arg Glu Arg Lys Arg Met His Asp Leu Asn Ile Ala Met
115 120 125
Asp Gly Leu Arg Glu Val Met Pro Tyr Ala His Gly Pro Ser Val Arg
130 135 140
Lys Leu Ser Lys Ile Ala Thr Leu Leu Leu Ala Arg Asn Tyr Ile Leu
145 150 155 160
Met Leu Thr Asn Ser Leu Glu Glu Met Lys Arg Leu Val Ser Glu Ile
165 170 175
Tyr Gly Gly His His Ala Gly Phe His Pro Ser Ala Cys Gly Gly Leu
180 185 190
Ala His Ser Ala Pro Leu Pro Ala Ala Thr Ala His Pro Ala Ala Ala
195 200 205
Ala His Ala Ala His His Pro Ala Val His His Pro Ile Leu Pro Pro
210 215 220
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Val Ser Ser
225 230 235 240
Ala Ser Leu Pro Gly Ser Gly Leu Pro Ser Val Gly Ser Ile Arg Pro
245 250 255
Pro His Gly Leu Leu Lys Ser Pro Ser Ala Ala Ala Ala Ala Pro Leu
260 265 270
Gly Gly Gly Gly Gly Gly Ser Gly Ala Ser Gly Gly Phe Gln His Trp
275 280 285
Gly Gly Met Pro Cys Pro Cys Ser Met Cys Gln Val Pro Pro Pro His
290 295 300
His His Val Ser Ala Met Gly Ala Gly Ser Leu Pro Arg Leu Thr Ser
305 310 315 320
Asp Ala Lys
Claims (12)
- 뇌조직을 추출하는 단계;
콜라겐 및 피브리노겐을 포함하는 용액, 트롬빈 및 상기 뇌조직을 혼합하여 뇌조직이 포함된 하이드로겔을 준비하는 단계;
ROCK 억제제를 포함하는 배양 배지를 이용하여 2일 이상 7일 이하의 기간 동안 뇌조직을 배양하는 단계;
상기 배양된 뇌조직을 이용하여 콜라겐 하이드로겔에서 신경줄기세포를 증폭배양하는 단계;
콜라게나제를 포함하는 시트 용해제를 이용하여 증폭배양된 신경줄기세포를 분리하는 단계; 및
분리된 신경줄기세포를 배양 접시에서 배양하는 단계;를 포함하며,
상기 신경줄기세포는 Tuj-1(Neuron-specific class III beta-tubulin)에 대해 음성인 면역학적 특성을 갖고, Olig2(Oligodendrocyte transcription factor 2)에 대해 양성인 면역학적 특성을 갖고, SOX2, PAX6, DCX 및 Olig2로 이루어진 군에서 선택된 하나 이상의 마커를 발현하는 것인, 신경줄기세포 배양 방법.
- 삭제
- 제1항에 있어서,
상기 하이드로겔 100 중량부에 대하여, 상기 콜라겐은 2 중량부 이하로 포함되는 것인, 신경줄기세포 배양 방법.
- 삭제
- 삭제
- 삭제
- 삭제
- 제1항에 있어서,
상기 시트 용해제는 상기 시트 용해제 100 중량부에 대하여 콜라게나제를 0.01 중량부 이상 1 중량부 이하로 포함하는 것인, 신경줄기세포 배양 방법.
- 삭제
- 제1항에 있어서,
상기 신경세포를 동결보존하는 단계를 더 포함하는 것인, 신경줄기세포 배양 방법.
- 삭제
- 삭제
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