이 작품은 Det에 FRIE Forskningsråd / Sundhed OG Sygdom에서 유전학 파일럿 상에 NCCR 프론티어, 청소년 당뇨병 연구 재단의 41-2009-775 그랜트 12-126875 순차적으로 투자되었다. 저자는 비디오 촬영을 호스팅 스파 놀리 실험실 감사합니다.

이 프로토콜은 쥐 E10.5에서 파생 된 췌장 organoids 상피 췌장 세포를 해리 성장하는 것을 목표로하고있다. 이 프로토콜은 동물 실험에 대한 윤리적 승인을 필요로합니다. E10.5 마우스 배아에서 지느러미 췌장 버드 1. 해부 <ol><li> 가위로 복부를 열고 배아 일 (E) 10.5 초과 임신 쥐를 희생, 두 자궁 뿔을 제거하고 차가운 인산 완충 생리 식염수 (PBS) 또는 둘 베코 가득 10cm 페트리 접시에 배치 수정 필수적인 매체 (DMEM)을 얼음에 보관. 세포 파종에 희생에서 전체 실험은 세포 손상을 방지하기 위해 60 ~ 90 분에 이루어집니다. </li><li> 작은 가위를 사용하여 개별 배아 세그먼트로 자궁을 분리합니다. 차가운 PBS와 35mm 페트리 접시에 하나의 배아를 전송하고 위의 조명과 해부 현미경을 시각화. 해부 집게와 주변 근육, 탈락과 노른자 SA를 제거C와 배아를 노출합니다. 다시 차가운 DMEM 매체에 배아를 놓습니다. 태아 간단 3 ㎖ 플라스틱 전송 피펫 / 적기를 사용하여 전송 될 수있다. 계속하기 전에 유사한 방식으로 모든 개인 배아를 분리합니다. </li><li> PBS에 깨끗한 35mm 페트리 접시에 배아를 놓습니다. <ol><li> 앞다리를 제거하는 얇은 집게 (0.05 mm 폭)를 사용합니다. 조심스럽게 척수 지역에서 소화 기관 (그림 1)을 분리 개방에 집게를 삽입합니다. 선택 사항 : 더 편리하게, 위를 참조하십시오 아래 노른자 줄기 아래 마음과 꼬리 영역으로, 태아의 상체를 제거합니다. </li><li> 위, 간, 소장을 찾습니다. 집게를 사용하여 소장에 위장에서 소화 기관을 분리하고 (그림 1) 얼음에 차가운 DMEM에 배치합니다. 지느러미 췌장 꽃 봉오리는 위장 등쪽, 후부 (그림 1)가 포함되어 있습니다. </li><li> 시미에있는 모든 개인 위장관 분리계속하기 전에 고맙다 방식. 배아 유전자형을해야 할 경우, 절개시에 꼬리를 수집하고 얼음에 차가운 DMEM과 24 웰 플레이트에서 다른 잘되는 모든 개인 위장관을 유지합니다. </li></ol></li><li> 차가운 PBS에 깨끗한 35mm 페트리 접시에 한 위장관 놓습니다. 이제 해부 현미경 등의 췌장 봉오리를 시각화하고 해부 시야에서 아래에서 조명을 사용합니다. 이러한 조건은 볼륨의 시각화를 최적화합니다. 전해 날카롭게 텅스텐 바늘 또는 20 G 주사기 바늘을 사용하여, 상피 주위 가급적 간엽 가진 췌장 새싹 (도 1)을 격리. <ol><li> 2 ~ 3 분 동안 차가운 디스 파제 용액 (1.25 ㎎ / ㎖)를 포함하는 배양 접시에 고립 된 꽃 봉오리를 전송합니다. 이 시점에서 전송이 가장 편리하게 입 통제 유연한 튜브에 부착 불꽃 뽑아 50 μL 유리 모세관을 수행 할 수 있습니다. 대안 적으로, 그러나 더 RI 함께새싹을 잃을 SK는 적절한 플라스틱 팁을 10 μL 자동 피펫 (Pipetman)를 사용합니다. </li><li> 미세한 제어 췌장 봉오리 흡입과 배출을 수행합니다. 다시 PBS에서 췌장 봉오리를 넣어. 또한 유리 모세관 (그림 1)를 사용하여 바늘과 부드러운 열망을 가진 중간 엽에서 분리 된 췌장 봉오리를 청소합니다. </li><li> 전체 중간 엽을 제거 할 때, 차가운 PBS에서 췌장 싹 씻어; (60 - 웰 미니 트레이 차가운 DMEM 10 μL로 가득) 개인 우물에서 차가운 DMEM 각 봉오리를 전송합니다. 그것은 조직이 매우 끈적하게하는, 디스 파제에서 중간 엽을 제거하지 않는 것이 중요합니다. </li></ol></li></ol> 분산 된 세포의 2. 도금 및 문화 <ol><li> 세정 10 ㎕의 PBS로 가득 60도 미니 트레이의 원추형의 우물에 불꽃 뽑아 유리 모세관 모든 배아에서 해부 상피를 전송합니다. <ol><li> 트립신 0.05 %의 10 μL에 꽃 봉오리로 이동ND는 4 분 동안 37 ° C에서 배양 할 수 있습니다. 10 μL의 DMEM + 10 % 소 태아 혈청 (FCS)와 우물에 새싹 전송하여 트립신을 불 활성화. </li><li> 피펫 풀러으로 뽑아가는 모세 혈관을 통해 흡인 세포 현탁액을 떼어 놓다. 이것은 세포를 해리 상하 피펫 팅하면서이 단계에서 기포를 방지하는 것이 중요하다. 췌장 organoids 최적 ~ 15 세포의 작은 그룹에서 시작하기 때문에 부분적인 분리는 (그림 1)를 추천합니다. </li></ol></li><li> 개별 처리로 인한 차이를 최소화하기 위해 에펜 도르프 튜브에 몇개의 배아 세포를 해변. 1:3 비율로 냉장 마트 리겔에서 세포 현탁액을 희석. 나누어지는 96 - 웰 플레이트, 8 μL / 잘 또는 영상에 최적화 된 접시에 (아래 참조)이 혼합물. </li><li> 리겔은 두껍게 할 수 있도록 5 분 동안 37 ° C에서 접시를 품어. 선택의 매체의 70 μL로 우물을 채우기 (organoid 또는 구, 탭보기 참조레 1, 2), 37 ° C에서 5 % CO 2, 95 %의 공기를 포함하는 습한 환경에서 떠나 </li><li> 매 4 일째 매체를 교체합니다. 매일 성장하는 췌장 식민지를 모니터링 및 이미징하여 프로세스를 문서화합니다. </li><li> 이전에보고 된 28 또한 작은 분자 또는 단백질, 간섭 실험을 위해이 단계에서 배지에 첨가 할 수있다. </li></ol> 표 1 : Organoid 매체. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 소재의 이름 </td><td> 주식 집중 </td><td> 최종 매체에 집중 </td><td> 주식의 볼륨 </td></tr><tr><td> 페니실린 - 스트렙토 마이신 </td><td> 100 % </td><td> 1 % </td><td> 50 μL </td></tr><tr><td> 녹아웃 혈청 교체 (보충) </td><td> 100 % </td><td> 10 % </td><td> 500 μL </td></tr><tr><td> 2 - 머 캅토 에탄올 </td><td> 14.3 M </TD&gt; <td> 0.1 ㎜ </td><td> 1 μL </td></tr><tr><td> 포르 미리 스테이트 아세테이트 (PMA) </td><td> 16 μM </td><td> 16 nM의 </td><td> 5 μL </td></tr><tr><td> Y-27632 (ROCK 저해제) </td><td> 50 mM의 </td><td> 10 μM </td><td> 1 μL </td></tr><tr><td> EGF </td><td> 50 ㎍ / ㎖의 </td><td> 25 NG / ML </td><td> 2.5 ㎕의 </td></tr><tr><td> 재조합 인간 1 R-spondin </td><td> 250 ㎍ / ㎖의 </td><td> 500 NG / ML </td><td> 10 μL </td></tr><tr><td> - 또는 - </td><td></td><td></td><td></td></tr><tr><td> 재조합 마우스 R-spondin 1 </td><td> 250 ㎍ / ㎖의 </td><td> 500 NG / ML </td><td> 10 μL </td></tr><tr><td> 재조합 인간 FGF1 (aFGF) </td><td> 100 ㎍ / ㎖의 </td><td> 25 ㎍ / ㎖의 </td><td> 1.25 μL </td></tr><tr><td> 헤파린 (Liquemin) </td><td> 2500 U / ㎖ </td><td> 2.5 U / ㎖ </td><td> 2 μL </td></tr><tr><td> 권장binant 인간 FGF10 </td><td> 100 ㎍ / ㎖의 </td><td> 100 NG / ML </td><td> 5 μL </td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4,412.25 μL </td></tr><tr><td> 합계 </td><td></td><td></td><td> 5,000 μL </td></tr></tbody></table> 표 2 : 구 매체. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 소재의 이름 </td><td> 주식 집중 </td><td> 최종 매체에 집중 </td><td> 주식의 볼륨 </td></tr><tr><td> 페니실린 - 스트렙토 마이신 </td><td> 100 % </td><td> 1 % </td><td> 50 μL </td></tr><tr><td> B27의 X50 (보충) </td><td> 100 % </td><td> 10 % </td><td> 100 μL </td></tr><tr><td> 재조합 인간 FGF2 (bFGF를) </td><td> 100 ㎍ / ㎖의 </td><td> 64 NG / ML </td><td> 3.2 ㎕의 </td></tr><tr><td> Y-27632 (ROCK 저해제) </td><td> 50 mM의 </td><td> 10 μM </td><td> 1 μL </td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4845.8 μL </td></tr><tr><td> 합계 </td><td></td><td></td><td> 5000 μL </td></tr></tbody></table> Organoid 개발의 진행 상황 3. 영상 <ol><li> 이미지 organoids 하나를 매일 또는 시간 경과 현미경 형광 시간 경과 현미경을 사용하여. 시간 경과 영상의 경우, XY (Z) 자동 거꾸로 형광 현미경을 사용합니다. </li><li> 4 - 웰 플레이트 또는 2-5 ML 매체로 채워진 유리 바닥 접시에 3 μL의 예금 작은 방울. 배 장거리 목적으로 이미지. 참고 : 형광 트레이서을 표현 형질 전환 마우스를 사용할 수 있습니다. 예를 들어 영화 1은 Pdx1 - Ngn3-ER TM-IRES-nGFP + 마우스 4에서 organoids의 초기 확장. 핵 GFP 개별 개체 만 유사한 원리는 형광 막과 세포를 추적하는 데 적용 할 수있는 세포를 추적 할 수 있도록 사용자가 그러한MT / MG 마우스 29로. </li><li> 초점이 감도을 방지하고 수동으로 정의 된 위치에 3 일 이상 1 사진 / 시간을 취할 수있는 소프트웨어 제어 자동화를 설정하는 세포를 파종 한 후 시간 경과 영상 3 시간을 시작합니다. 각 위치에 대한, 형광 마커의 발현을보고, 미분 간섭 대비 (DIC) 이미지뿐만 아니라, GFP 신호를 획득. </li></ol> 조직학에 대한 Organoids 4. 복구 <ol><li> 얼음에 96 - 웰 플레이트를 배치하고 얼음 차가운 PBS로 교체, 매체를 제거합니다. 이것은 부분적으로 리겔을 해중합. </li><li> 조심스럽게 전체 아키텍처를 방해하지하기 위해 1000 μL 팁을 사용하여 주변의 리겔을 제거, 각 organoid를 대기음. 얼음 차가운 PBS로 잘 각 organoid을 전송합니다. 얼음에 플레이트를 유지합니다. 리겔 직접 고정도 가능합니다. </li><li> 4 % 파라 포름 알데히드 (PFA)로 15 분 동안 organoid 고쳐 자당에 냉동 보존 및 젤라틴에 포함. 각 처리organoid 이전 (요한슨 등., 2007) 4 기술로 저온 절편 제작 및 조직학에 대한. </li></ol> PCR 및 생화학을위한 Organoids 5. 복구 <ol><li> 얼음에 96 - 웰 플레이트를 놓고 매체를 제거합니다. 세포의 RNA를 안정시키고 보호하기 위해 잘 당 RNAlater의 60 μl를 추가합니다. </li><li> 부분적으로 얼음을 해중합하여 각 아니라 기계적에서 젤을 방해. 전체 아키텍처를 중단 또는 200 μL 팁으로 기계적으로 젤을 방해하여 (리겔 포함) 전체를 잘 복구 할 수 있도록 1000 μL 팁을 사용하여 개별 organoids를 복구 하나. 이는 RNAse 무료 끈적 에펜 도르프 튜브에 잘 콘텐츠를 전송하기 위해 1000 μL 팁을 사용하여 얼음에 보관. </li><li> 60 μL의 RNAlater에 우물을 세척하고 같은 에펜 도르프 튜브에 남아있는 내용을 추가합니다. </li><li> 4 ℃에서 500 ~ 1,000 XG에서 5 분 튜브를 스핀 </li><li> 만 20 ~ 30 μl를 떠나, 뜨는을 제거함께 펠릿과 튜브 RNAlater의. 생화학의 경​​우, 가능한 한 건조 샘플을 저장합니다. </li><li> 즉시 RNAlater에서 샘플을 동결하지 마십시오; 4 ° CO / N에 상점 (RNAlater 철저하게 조직을 침투 할 수 있도록). 조직은 장기 저장을 위해 -20 ° C에 저장할 수 있습니다 나중에 조직의 파괴와 RNA의 소량의 추출을 위해 처리 할 수​​ 있습니다. </li></ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+identification+of+multipotent+precursors+from+adult+mouse+pancreas+that+generate+neural+and+pancreatic+lineages.">Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages.</a> Nat Biotechnol. 22, 1115-1124 (2004).</li><li>Jin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colony-forming+cells+in+the+adult+mouse+pancreas+are+expandable+in+Matrigel+and+form+endocrine/acinar+colonies+in+laminin+hydrogel.">Colony-forming cells in the adult mouse pancreas are expandable in Matrigel and form endocrine/acinar colonies in laminin hydrogel.</a> Proc Natl Acad Sci U S A. 110, 3907-3912 (2013).</li><li>Sugiyama, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituting+pancreas+development+from+purified+progenitor+cells+reveals+genes+essential+for+islet+differentiation.">Reconstituting pancreas development from purified progenitor cells reveals genes essential for islet differentiation.</a> Proc Natl Acad Sci U S A. 110, 12691-12696 (2013).</li><li>Huch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unlimited+in+vitro+expansion+of+adult+bi-potent+pancreas+progenitors+through+the+Lgr5/R-spondin+axis.">Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.</a> Embo J. , (2013).</li><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. , (2009).</li><li>Ootani, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+in+vitro+intestinal+epithelial+culture+within+a+Wnt-dependent+stem+cell+niche.">Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche.</a> Nat Med. 15, 701-706 (2009).</li><li>Barker, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lgr5(+ve)+stem+cells+drive+self-renewal+in+the+stomach+and+build+long-lived+gastric+units+in+vitro.">Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro.</a> Cell Stem Cell. 6, 25-36 (2010).</li><li>Huch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+expansion+of+single+Lgr5++liver+stem+cells+induced+by+Wnt-driven+regeneration.">In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration.</a> Nature. 494, 247-250 (2013).</li><li>Lukacs, R. U., Goldstein, A. S., Lawson, D. A., Cheng, D., Witte, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+cultivation+and+characterization+of+adult+murine+prostate+stem+cells.">Isolation, cultivation and characterization of adult murine prostate stem cells.</a> Nat Protoc. 5, 702-713 (2010).</li><li>Rock, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+cells+as+stem+cells+of+the+mouse+trachea+and+human+airway+epithelium.">Basal cells as stem cells of the mouse trachea and human airway epithelium.</a> Proc Natl Acad Sci U S A. 106, 12771-12775 (2009).</li><li>Eiraku, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organizing+optic-cup+morphogenesis+in+three-dimensional+culture.">Self-organizing optic-cup morphogenesis in three-dimensional culture.</a> Nature. 472, 51-56 (2011).</li><li>Spence, J. 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D., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+double-fluorescent+Cre+reporter+mouse.">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45, 593-605 (2007).</li></ol>

The authors have nothing to disclose.

시험 관내에서 기능적인 베타 세포의 대규모 생산은 여전히 하나 비효율적이다. 이 도전적인 상황에서, 발달 생물학 연구 기능의 베타 세포의 분화에 필요한 정확한 신호를 해독하는 데 도움이 될 수 있습니다. 이 프로토콜은 체외에서 배아의 췌장 전구 세포의 유지 보수, 확장과 차별화 할 수 있습니다. 이것은 다른 내분비 호르몬을 공동으로 표현하지 않는 인슐린을 생산?...

장기 문화는 생체 내 연구에서 복잡하지만 관련성이 높은 사이의 간격 및 세포 라인 모델의 편리하지만 대략적인 시뮬레이션을 다리에 유용한 모델을 제공합니다. 췌장의 경우, 내분비와 외분비 세포 시뮬레이션 세포주가 변형 되더라도 췌장 전구 세포로 완전히 동등한 어떠한 세포주도 없다. 성인 전체 췌장을 배양 할 수 없습니다; 절연 내분비 아일렛 세포 증식없이 몇 주 동안 유지 될 수 있으며, 조직 조각은 몇 시간에서 5 시험 관내에서 유지 될 수있다. 배아의 췌장 문화가 널리 개발을 연구뿐만 아니라 사용되었습니다뿐만 아니라 8을 처리하는 이미지로, 상피 - 간엽 상호 작용 4,6,7을 조사하기 위해 화학적으로 그 9 방해 할 수 있습니다. 두 기관 배양 방법이 주로 사용된다 : 첫 번째는 conve입니다 피브로넥틴 코팅 플레이트 2에 췌장 꽃 봉오리를 배양에 구성촬상 목적 더욱 편리 할; 두 번째 옵션은 문화에 가장 형태 형성을 유지 공기 - 액체 인터페이스 3,4에서 필터에 대한 기관입니다. 매우 유용하지만, 이러한 방법은 평탄화의 어느 정도 이어질; 정상적인 발달에 비해 시작 인구가 췌장 세포 및 중간 엽 세포의 모든 유형을 포함하는 복잡한 같이 선조의 팽창은 매우 제한된다. 문화 능력과 분산 일차 전지를 확장 계보 관계를 연구하고 고립 된 세포 유형 (10)의 고유 특성을 발견 할 가치가있다. 스기야마 등. 11 췌장 전구 세포 및 피더 레이어에 문화에 3 ~ 5 일 일부 기능 문자를 유지 내분비 전구 세포를 유지할 수 있습니다. 조상의 자연 / 줄기 세포하지만 neurosphere를 12, 13 mammospheres에 가깝다 Pancreatospheres는 성인 독도 및 유관 세포에서 확장되었습니다이러한 분야를 생성하는 것은 명확하지 않다. 또, 생리 현상과는 대조적으로, pancreatospheres 일부 뉴런에게 14,15을 함유 하였다. 분야는 최근 배아 췌장 전구 세포 (16, 17)과 좋은 시조 확장 이후의 분화와 pancreata 18 재생에서 생산하지만, 형태 형성을 요점을 되풀이하는 데 실패했다. 소형 기관에 자체 조직으로 분산 종종 정의 세포에서 3D 모델은 최근 번성하고 소장 (19, 20), 배 (21), 간 (22), 전립선 (23)과 기관 등의 여러 장기의 개발 또는 성인 매출을 시뮬레이션 한 24. 광섬유 컵 25, 26 또는 뇌 부위 (27)의 경우와 같이 몇몇 경우에, 형태 형성 발달과 분화는, ES 세포로부터 3D로 효과적으로 요약되었다. 여기, 우리 DES그들은 차별화 자체 구성 할 수 있습니다 3D 리겔 비계에서 해리 다 능성 췌장 전구 세포를 확장하는 방법을 cribe.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>KnockOut Serum replacement (supplement)</td>
			<td>Gibco</td>
			<td>10828-028</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>3148-25ML</td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>Phorbol Myristate Acetate (PMA)</td>
			<td>Calbiotech</td>
			<td>524400-1MG</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK inhibitor)</td>
			<td>Sigma Aldrich</td>
			<td>ab120129</td>
			<td>Stock keept at -20&#176;C- Attention! Stability/source is a frequent source of problems</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Sigma Aldrich</td>
			<td>E9644-2MG</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human R-spondin 1</td>
			<td>R&#38;D</td>
			<td>4645-RS-025/CF</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>&#160;- or -&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse R-spondin 1</td>
			<td>R&#38;D</td>
			<td>3474-RS-050</td>
			<td>Stock keept at -80&#176;C</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF1 (aFGF)</td>
			<td>R&#38;D</td>
			<td>232-FA-025</td>
			<td>Stock keept at -80&#176;C- do not include to increase beta cell production</td>
		</tr>
		<tr>
			<td>Heparin (Liquemin)</td>
			<td>Drossapharm</td>
			<td></td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF10</td>
			<td>R&#38;D</td>
			<td>345-FG-025</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>21331-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>B27 x50 (supplement)</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF2 (bFGF)</td>
			<td>R&#38;D</td>
			<td>233-FB-025</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK inhibitor)</td>
			<td>Sigma Aldrich</td>
			<td>ab120129</td>
			<td>Stock keept at -20&#176;C- Attention! Stability/source is a frequent source of problems</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>21331-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Trypsin 0.05%</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>RNAlater - RNA stabilizing reagent</td>
			<td>Qiagen</td>
			<td>76104</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>Dispase&#160;</td>
			<td>Sigma Aldrich</td>
			<td>D4818-2MG</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>BSA for reconstitution</td>
			<td>Milipore</td>
			<td><a href="http://www.millipore.com/catalogue/item/81-068-3">81-068</a></td>
			<td>For reconstituition of cytokines&#160; - Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Gibco</td>
			<td>16141079</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>60 well MicroWell trays</td>
			<td>Sigma Aldrich</td>
			<td>M0815-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well plates</td>
			<td>Thermo Scientific</td>
			<td>176740</td>
			<td></td>
		</tr>
		<tr>
			<td>95-well plates F bottom</td>
			<td>Greiner Bio</td>
			<td>6555180</td>
			<td></td>
		</tr>
		<tr>
			<td>Glas bottom plates</td>
			<td>Ibidi</td>
			<td>81158</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposal micropittes</td>
			<td>Blaubrand</td>
			<td>708745</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro pancreas organogenesis from dispersed mouse embryonic progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

3D 리겔에서 해리와 시드 E10.5 지느러미 췌장 전구 세포는 췌장 개발을 요점을 되풀이하다. 조상은 쉽게 형광 기자와 올 수 있습니다. 우리의 경우 우리는 (그림 2) Neurog3 4 활성화하지 않고 타목시펜의 부재에 따라서 Pdx1 프로모터 (Pdx1 - Ngn3-ER TM-nGFP) (동영상 1)에 의해 제어되는 핵 GFP 단백질을 발현하는 형질 전환 마우스를 사용. organoid 매체?...

Watch this Scientific Journal Video about In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors at JoVE.com

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

이 프로토콜에 기재된 삼차원 배양법 분지 기관에 자신의 상당한 팽창, 분화 및 형태 발생을 포함한 분산 배아 마우스 췌장 전구 세포로부터 췌장 개발 되풀이되었습니다. 이 방법은 영상, 틈새 시장의 기능 장애 및 조작에 대한 의무입니다.

분산 된 마우스 배아 전구 세포에서 생체 췌장 기관 형성에

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

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Biology

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

13.7K 조회수.  Swiss Institute for Experimental Cancer Research.  이 프로토콜에 기재된 삼차원 배양법 분지 기관에 자신의 상당한 팽창, 분화 및 형태 발생을 포함한 분산 배아 마우스 췌장 전구 세포로부터 췌장 개발 되풀이되었습니다. 이 방법은 영상, 틈새 시장의 기능 장애 및 조작에 대한 의무입니다. 

비디오: In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

공중 드롭 방법을 사용하여 마우스 배아 줄기 (MES) 세포의 체외 차별에

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

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생물학

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

자기 공명 영상을위한 인간 배아 줄기 세포의 체외 라벨에

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

마우스 콘 Photoreceptors에서 단일 셀 석션 레코딩

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

생체내 이미징 및 난소암의 Orthotopic 마우스 모델에서 치료 트리 트먼트

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

골격근에서 Progenitors의 정화

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

위한 시스템<em&gt; 예 생체</em배아 췌장의&gt; 배양

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

유도 만능의 줄기 세포에서 파생 마우스의 생성

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

배아의 마우스 하트을 개발에있는 셀의 레테르를 붙이는 및 사출

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

마우스 췌장에서 RNA 격리 : 리보 뉴 클레아 풍부한 조직

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

개발 배아 마우스 심장 밸브 지역에서 단백질 분리

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...