We gratefully acknowledge Dr. Deborah L. Gumucio as our advisor and for her invaluable support in defining the culture and imaging methods. We also thank Dr. Jim Brodie, Dr. Hong-Xiang Lu, Dr. Charlotte Mistretta, and Dr. Ann Grosse for their contributions to the development of the whole intestine organ culture system. Helpful discussions on imaging provided excellent advice from Dr. Chip Montrose, Michael Czerwinski and Sasha Meshinchi. All imaging was performed in the Microscopy and Image Analysis Laboratory at the University of Michigan. Funding support was provided by NIH R01 DK065850.

참고 : 모든 쥐가 실험 동물 의학 미시간 의과 대학 단위의 대학의 승인을 동물의 사용 및 관리에 대학위원회의 지침에 따라 프로토콜을 사용하여 인도적으로 처리 하였다. 1 전체 장기 문화 시스템 <ol><li> 미디어 및 문화 판의 제조 <ol><li> 조직 배양 후드에서 스톡 병 BGJb 매체 5 mL를 제거하고, 펜 / 스트렙토 5 mL를 추가한다. 5 밀리그램 / (PEN / 스트렙토)와 BGJb 매체의 49 ml의 아스코르브 산 1 ㎖를 첨가하여, 50 ㎖의 원뿔형 튜브에 미디어 콘텐츠의 작업을 준비한다. </li><li> 트랜스 웰 아래의 각각 1 ㎖에 첨가하여 6 웰 배양 플레이트를 준비한다. 참고 : 모든 6 트랜스 웰을 사용하지 않을 경우, 나중에 사용하기 위해 신선한 무균 6 웰 플레이트에 추가 트랜스 웰을 이동합니다. 세 10cm 무균 배양 접시에 각각 BGJb 미디어를 작업의 3 ML을 추가합니다. </li></ol></li><li> 해부를위한 준비 <ol><li> 70 % etha에서 해부 도구를 적시와 NOL 작업하는 동안 청소 도구를 보관하십시오. </li><li> 큰 얼음 양동이 해부 영역을 설정하고 6 웰 배양 판과 얼음에 BGJb 미디어와 10cm 배양 접시를 놓습니다. 해부와 문화에 대한 장을 준비하는 동안 가능한 한 많은 얼음에 작업 할 수 있습니다. </li><li> 태아 창자 수확 용 자궁 전위에 의해 이소 플루오 란 (100 μL) 이전 euthanization에 함께 실에서 성인 여성 생쥐를 마취. </li><li> 자신의 어머니로부터 태아를 수확 한 후, 각각의 태아가 신중하게 차트 13을 준비 Theiler에 따라 무대. 정기적으로 하나의 쓰레기 내에서 관찰되는 발달 단계에서 최대 24 시간의 변화에​​ 각 태아의 나이를 결정하는 포스트 성교 일에 의존하지 마십시오. </li></ol></li><li> 태아 내장의 해부 <ol><li> 창자를 제거하기 전에 잘린 태아를 안락사. </li><li> 괞 찮아 후 1 배 둘 베코 인산염 완충 식염수 (DPBS)의 각 장을 해부BGJb 미디어 작업과 10cm 페트리 접시로 CE. </li><li> 조심스럽게 배에서 비장과 위장과 상부 십이지장에서 췌장을 제거합니다. </li><li> 부드럽게 소장은 곧게 할 수 있도록 가능한 한만큼의 연결을 분리 한 많이 붙어있는 대망을 떠나 돌보는 대망 구분합니다. 참고 : 부드럽게 내강 내용이 소장을 통과하고 절단 끝에서 추방 할 수 있도록 세 동일한 세그먼트로 장을 분리 장 E14.5보다 나이 또는 사람들은 더 이상 24 시간 이상 배양하고 들어. 그러나, 배양을 위해 E13.5에 앞서, 장막 제대로 소장의 길이에 걸쳐 확장 할 수 있도록 부위의 3 세그먼트를 분리하기 전에 배양 한 후 24 시간까지 기다린 시작했다. </li><li> 문화 판 (1.1.2)의 트랜스 웰에 장 조각을 전송하기 위해 입 피펫을 사용합니다. </li><li> 그들은 transwel에 걸쳐 직선이되도록 필요에 따라 조심스럽게 내장의 위치를리터. 주 : 창자가 튜브를 통해 내강 내용을 이동 시작하면 장에있는 꼬임 장에 백업 및 손상의 원인이됩니다. </li></ol></li><li> 문화 및 치료 <ol><li> , 트랜스 웰 아래에서 용지를 제거 트랜스 웰에서 (추가 재조합 단백질이나 약물 억제제와 미디어 작업) 치료 미디어 700 μl를 추가합니다. </li><li> 내장의 상단에 치료 미디어 적가의 300 μl를 추가하고 트랜스 웰을 흡수 할 수 있습니다. 필요에 따라 내장 위치를​​ 조정합니다. </li><li> 치료 시약의 반감기에 따라 적어도 하루에 한 번 또는 더 자주 치료 미디어를 변경합니다. </li></ol></li><li> 치료 시약의 지역화 된 소스 단백질 젖 아가 로스 비드의 제조 <ol><li> 스토리지 컨테이너에 아가로 오스 비즈를 재현 탁하고, 1.5 ml의 미세 원심 분리 튜브에 아가 로스 비드의 100 μl를 전송합니다. </li><li> 멸균 1X pH가 튜브의 남은 볼륨을 채우기osphate 완충 식염수 (PBS)와 그들을 씻어 구슬을 섞는다. 몇 분 구슬이 바닥에 정착 한 후 구슬의 상단에에서 PBS를 피펫 할 수 있도록하는 랙에 미세 원심 분리 튜브를 놓습니다. 멸균 1X PBS와 미세 원심 분리 튜브를 채우고 두 번 더 세척 과정을 반복합니다. </li><li> 두 번 치료를 원하는 농도로 관심의 단백질을 준비합니다. </li><li> 배 단백질 주식의 5 μL로 세척 아가로 오스 비드 5 μl를 혼합하고 부드럽게 단백질 구슬을 선동. 부드럽게 매 10 ~ 15 분을 혼합, 1 시간 동안 얼음에 튜브를 놓습니다. </li><li> 장내에 구슬을 배치하기 전에 멸균 1X PBS에 잠시 구슬을 씻어. </li></ol></li><li> 아가로 오스 구슬의 배치 <ol><li> 부드럽게 당겨 모세관 바늘 입 피펫을 사용하여 내장의 상단에있는 아가로 오스 비드를 배치합니다. 거친 취급 장에 손상을 부상당한 지역에 융모 발달을 차단하므로 매우주의 구슬을 놓습니다. </li><li> 창자의 연동 운동과 배양 시간이 지남에 따라 내장의 롤 오프한다 배치 된 구슬의 절반 정도를 받아야하기 때문에 분석에 필요한 두 배나 많은 구슬을 놓습니다. </li></ol></li><li> 배양 내장의 고정 <ol><li> 조심스럽게 트랜스 웰 아래에서 치료 용지를 제거하고 3 분 동안 건조한 공기에 내장 할 수 있습니다. </li><li> 다음, 정착 시간의 나머지 정착액 2 ㎖와 소장의 상부 커버, 2 분 동안 트랜스 웰 아래 정착액 1 ㎖를 추가한다. 4 ° C 또는 실온에서 2 시간 동안 4 % 파라 포름 알데히드 O / N으로 수정합니다. </li></ol></li></ol> 고정 창자의 2 이미징 <ol><li> Wholemount 영상 <ol><li> 1X DPBS 100 ㎕와 슬라이드 (내생​​ 형광 포함) 전체 창자를 놓습니다. 부드럽게 장을 마련하기 위해 집게를 사용합니다. </li><li> 조직 개월을주의 깊게 70 % 글리세롤 100 μl를 DPBS를 제거하고 즉시 추가 실험실 조직을 사용하여투명 재 및 이미징의 가능한 깊이를 증가시킨다. NOTE : 소장의 상기 관통 원하는 경우, 대신에 70 % 글리세롤에 실장 부위에, 10 ~ 30 분 동안 초점 클리어 용액 몇 방울의 소장 소크. 초점 클리어 솔루션을 피펫 및 마운트 클리어와 장을 마운트합니다. </li><li> 모델링 점토로 커버 슬립의 네 모서리를 각각 담궈 부위를 평탄화로부터 커버 슬립을 유지하는 슬라이드 및 커버 슬립 사이에 스페이서로서 사용하는 점토를 소량 픽업. </li><li> 면봉으로 적용 녹은 VALAP와 슬라이드에 커버 슬립을 밀봉합니다. </li><li> 이미지 중 하나를 세우거나 거꾸로 공 초점 현미경에 내장. 자세한 내용은 설명을 참조하십시오. </li></ol></li><li> (장 구조의 단면도에 대한) 고정 장의 Vibratome의 절편 <ol><li> 작은 플라스틱 금형 7 % 아가로 오스에 포함 된 장에 (10 × 10 × 15mm) 허용 아가로 오스 블록냉각 및 응고. </li><li> 순간 접착제와 vibratome 수집 단계에 플라스틱 금형 및 접착제 아가로 오스 블록에서 아가로 오스 블록을 제거합니다. </li><li> 얼음 차가운 1X DPBS와 컬렉션 무대를 채 웁니다. </li><li> 100 ~ 150 μm의 사이에 두께 부분을 잘라. 두꺼운 부분은 단면에 깊은 시각화 (2.1.2 참조) 청산 솔루션을 사용하십시오. </li><li> 4 ° C에서 저장을위한 6 웰 플레이트의 우물에 수집 단계부터 vibratome 섹션을 붓는다. </li></ol></li><li> 고정 vibratome 단면 조직의 면역 형광 염색 <ol><li> 1X DPBS와 함께 24 웰 플레이트에 잘 당 5-12 vibratome 섹션을 배치합니다. </li><li> 1X DPBS를 제거하고 잘 당 permeabilization 용액 500 μl를 추가합니다. 부드럽게 실온에서 25 분 동안 샘플을 바위. </li><li> permeabilization 후 1X PBS로 샘플을 3 회 씻는다. </li><li> 차단 솔루션의 500 μL에 적어도 30 분 동안 샘플을 차단합니다. </li><li> 차단, 전자 후차단 솔루션에 희석 차 항체의 500 μL와 차단 솔루션을 체인지와 4 ° CO / N에서 샘플을 유지합니다. 참고 : 냉동 식품 및 파라핀 섹션에서 잘 작동 차 항체 희석은 일반적으로 그러나 일반적으로 프로토콜 (예를 들어, BrdU의) 잘 작동하지 않는 핵 항원 검색을 필요로하는 항체도 vibratome 섹션에서 잘 작동합니다. </li><li> 다음 일, 차단 솔루션으로 15 분 동안 샘플 때마다 세 번 씻는다. </li><li> 세안 후, 차단 솔루션에 희석 차 항체의 500 μl를 적용합니다. 실온에서 2 시간 45 분 동안 샘플을 빛으로부터 형광을 보호하고 바위에 알루미늄 호일에 24 웰 플레이트를 감싸. </li><li> 15 분마다 1X PBS로 샘플을 3 회 세척하고 2.4 절에 설명 된대로 슬라이드 vibratome 섹션을 탑재합니다. </li></ol></li><li> vibratome 섹션을 장착 <ol><li> vibratome 두 번 STI의 얇은 조각을 절단하여 장착 슬라이드를 준비CK 테이프와 슬라이드의 긴 측면을 따라 그들을 배치. </li><li> 조심스럽게 1X PBS 100 ㎕의 드롭 슬라이드에 두 번 스틱 테이프 사이에 5 ~ 10 vibratome 섹션을 배치합니다. </li><li> 모든 섹션을 펼치고 슬라이드에 걸쳐 단일 층으로 그들을 밖으로 확산. </li><li> 평면 앉아에서 커버 슬립을 방지 할 수 초과 아가로 오스 또는 고르지 않은 비트를 제거합니다. </li><li> 조심스럽게 vibratome 섹션 주위에서 초과 1X PBS를 흡수하기 위해 연구소 조직을 사용합니다. </li><li> vibratome 부 위에 장착 수성 매질의 드롭을 추가 한 후 커버 슬립. </li><li> VALAP가 면봉으로 적용 용융와 슬라이드로 된 커버를 밀봉. </li><li> 이미지 중 하나를 세우거나 거꾸로 공 초점 현미경에 vibratome 섹션. 영상 시스템의 선택에 대한 더 자세한 설명을 참조하십시오. </li></ol></li></ol> 전체 창자의 3 라이브 영상 창자 fetuse에서 수확연결된 대망가 그대로 유지와의 E12.5 후, 성공적으로 위의 시스템을 사용하는 문화에 융모을 개발한다. 따라서이 시스템은 생체 시간 경과 형태 형성의 3 차원 뷰를 위해 재구성 될 수있는 현상 장의 라이브 Z 단면 영상의 캡쳐를 허용한다. <ol><li> 라이브 영상 설정 <ol><li> 좋은 메쉬 화면으로 개별 폴리 카보네이트 막을 부착 순간 접착제를 사용합니다. 이 수직으로 공 초점 현미경의 침지 렌즈 아래 장소에 소장을 보유하는 지원 발판을 제공합니다. 라이브 영상 현미경의 선택에 대한 자세한 내용은 토론 섹션을 참조하십시오. </li><li> 중앙 잘 문화 판에 메쉬 스크린을 놓습니다. </li><li> 폴리 카보네이트 막에 해부 장을 놓고 양쪽 끝에 순간 접착제 한 방울을 추가 할 수 있습니다. 장을 부착시킬만큼의 접착제를 사용하지만 코트 그것! </li><li> 폴리 카보네이트 아래 페놀 레드의 문화 미디어 무료 추가중심도의 문화 판에서 막. </li><li> 습도를 제공하도록 배양 플레이트의 외연에 물을 첨가. </li><li> 이미징 동안 대장의 움직임을 제어하는​​ 미디어 3 ㎖에 1X PBS에서 자일 라진 (xylazine)의 다섯 솔루션 : 태아 소장은 문화 수축의 연동과 같은 파도를 겪는 때문에, 하나의 100 μl를 추가합니다. 이 투여 량은 융모 발달을 손상시키지 않고 약 8 ~ 10 시간 동안 장 움직임을 제어합니다. </li><li> 소장의 가로보기의 경우, 3 ~ 4 % 아가로 오스 솔루션의 라이브 창자를 포함하고 vibratome에 두꺼운 부분 (150 ~ 500 μm의) 잘라. 절단되는 소장의 강성 아가로 오스의 강성을 일치하고 경험적으로 발달 단계를 결정했다. 일반적으로, 3 % 아가로 오스 솔루션은 E14.5 미만의 창자에서 잘 작동하고 4 % 용액은 창자 E15-E16에 적합합니다. </li><li> (섹션 3.1.1에서와 같이) 메시 스크린에 부착 된 폴리 카보네이트 멤브레인 vibratome 섹션을 붙인문화는 제 3.1.2-3.1.6에서 설명. </li><li> 동조 테이블 직립 스탠드 이광자 형광 공 초점 현미경 (690-1,040 nm의 사이 여기 레이저)를 이용하여 실시간 이미징을 수행. </li><li> 이광자 레이저 GFP 신호 최상의 해상도 깊은 용입을 제공 내지 900에 동조. 이미징 동안은 조직의 손상을 줄일 수 있습니다. 또한, 가능한 최저로 발광 레이저의 파워를 설정하는 조직 손상을 줄여 준다. 통상적으로, GFP의 좋은 자극을 구하는 이광자 레이저에 12 % 전력을 사용한다. </li></ol></li></ol>

<ol>
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The authors have no financial conflicts to disclose.

동적 특성 및 개발 소장의 복잡한 조직 상호 작용은 이러한 형태 발생 이벤트의 전체 감사의 마음을 가지고 3D 시각화가 필요합니다. 이미징 기술 진화로, 능력 개발 / 개선과 함께, 기관 형성기 동안 공간 의사 소통과 상호 작용의 이해가 크게 향상되어 자세히 융모 형태 형성을 검사합니다. 전체 창자 배양 방법에 대해서도 테스트되었지만, 트랜스 웰 시스템은 긴 배양 시간?...

Each intestinal villus is composed of two main tissue compartments: an epithelial surface layer and a mesenchymal core. The mouse small intestine is formed at embryonic day 10 when a sheet of endoderm closes and seals to form a tube of epithelium surrounded by mesenchymal cells3. This flat tube of epithelium undergoes rapid proliferation, growing both in length and girth and undergoes dramatic rearrangements involving dynamic cell shape changes1. At the same time, the surrounding mesenchyme also undergoes many developmental processes including the formation of the vascular plexus, differentiation of smooth muscle and recruitment of enteric neurons4. In the proximal small intestine at embryonic day 14.5, condensations (clusters) of Hedgehog- and PDGF-responsive cells begin to form adjacent to the epithelium2,5. Formation of mesenchymal clusters continues to spread along the length of the intestine so that they cover the entirety of the small intestine by embryonic day 16.52. As mesenchymal clusters form, the epithelial cells closest to the clusters begin to withdraw from the cell cycle, while the other epithelial cells continue to proliferate. Those cells directly above the mesenchymal cluster that have withdrawn from the cell cycle begin to change shape as the emerging villus buckles into the lumen. Further growth of the villus is driven in part by the continued proliferation of the epithelium between the emerging villi. The mesenchymal clusters remain tightly adhered to the epithelium of the growing villus and continue to express a variety of signaling molecules. The wave of villus emergence propagates along the length of the small intestine following the formation of mesenchymal clusters. As the intestine continues to grow and the intervillus region extends between emerging villi, new mesenchymal clusters form adjacent to the intervillus epithelium and further rounds of villus emergence and growth ensue6.
Synchronized development of the epithelium and mesenchyme is essential for villus morphogenesis. Signaling molecules are secreted from one layer to the other where receptors receive and transduce the signal message in order to coordinate development between the epithelium and mesenchyme. Mesenchymal clusters act as signaling centers and express a variety of developmental morphogens7-10. Disruption of cluster formation or pattern results in loss of villus emergence and pattern. Inhibition of PDGF signaling results in fewer clusters and fewer villi and those villi that do form are misshapen following the abnormal clusters11. Loss of Hedgehog signaling results in complete loss of cluster formation and failure of villus emergence2,12. Together, these data demonstrate that clusters coordinate development of the villus epithelium with its mesenchymal core.
Using this whole organ culture system, we are able to alter signaling involved in epithelial-mesenchymal cluster cross-talk to determine the role of those signals in villus morphogenesis. Two-photon confocal optical sectioning and reconstruction afford the ability to visualize cluster formation and villus emergence in three-dimensions and better understand the spatial relationships between the mesenchymal clusters and their overlying epithelium. Extending the culture system to four dimensions, we can capture z-stacks of developing clusters and villi over time and observe these interactions. Ultimately, the ability to follow villus development in this manner and observe changes that occur with altered signaling will revolutionize the understanding of epithelial mesenchymal interactions in villus morphogenesis.

<table border="0" cellpadding="0" cellspacing="0" width="1308">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Fine dissecting forceps&#160;</td>
			<td style="width:223px;">Fine Science Tools</td>
			<td style="width:133px;">&#160;11254-20</td>
			<td style="width:444px;">2 pairs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Ethanol</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1x sterile Dulbecco&#39;s Phosphate-Buffered Saline (DPBS)</td>
			<td>Gibco&#160;</td>
			<td>14040-133</td>
			<td style="width:444px;">500 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well plates</td>
			<td>Costar</td>
			<td>3516</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well plates</td>
			<td>Costar&#160;</td>
			<td>3524</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 x 15 mm petri dishes</td>
			<td>Falcon</td>
			<td>&#160;451007</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transwell plates, 24 mm inserts, 8.0 mm polycarbonate membranes</td>
			<td>Corning Costar&#160;</td>
			<td>3428</td>
			<td style="width:444px;">6 inserts per plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BGJb media</td>
			<td>Invitrogen&#160;</td>
			<td>12591-038</td>
			<td style="width:444px;">500 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PenStrep (10,000U/ml Penicillin; 10,000 mg/ml Streptomycin)</td>
			<td>Gibco</td>
			<td>&#160;15140</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ascorbic Acid</td>
			<td>Sigma&#160;</td>
			<td>A0278</td>
			<td style="width:444px;">make 5 mg/ml stock, filter, aliquot and store at -20 &#176;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Mouth pipet (Drummond 1-15 inch aspirator tube assembly)</td>
			<td>Fisher</td>
			<td style="width:133px;">&#160;21-180-10</td>
			<td style="width:444px;">remove the aspirator assembly and replace it with a 1000 &#181;l pipet tip which acts as an adaptor to plug in a 6 inch glass Pasteur pipet.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 inch glass pasteur pipets</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose beads</td>
			<td>BioRad&#160;</td>
			<td>153-7301</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Capillary Tubes</td>
			<td>World Precision Instruments&#160;</td>
			<td>TW100F-4</td>
			<td style="width:444px;">pull to needles</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">4% Paraformaldehyde</td>
			<td></td>
			<td></td>
			<td style="width:444px;">made in 1 x PBS, pH to 7.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Live Imaging Materials</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td style="width:444px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Culture plates</td>
			<td>Falcon&#160;</td>
			<td>353037</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine mesh stainless steel screen</td>
			<td>purchase at hardware store</td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Polycarbonate membranes</td>
			<td>Thomas scientific&#160;</td>
			<td>4663H25</td>
			<td style="width:444px;">alternatively, cut Corning Costar 3428 membranes off of transwell supports</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Instant glue</td>
			<td>purchase at hardware store</td>
			<td style="width:133px;">gel based preferrably</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 x 10 mm plates</td>
			<td>Falcon&#160;</td>
			<td style="width:133px;">351008</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7% agarose</td>
			<td>Sigma</td>
			<td>&#160;A9414</td>
			<td style="width:444px;">prepare w/v in 1x DPBS, heating to dissolve in a waterbath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">minutien pins</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:133px;">26002-20</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenol red free media (DMEM)</td>
			<td>Gibco&#160;</td>
			<td style="width:133px;">21063-029</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine (100 mg/ml)</td>
			<td>AnaSed&#160;</td>
			<td style="width:133px;">139-236</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Matrigel</td>
			<td>BD</td>
			<td style="width:133px;">356231</td>
			<td style="width:444px;">basement membrane matrix, growth factor reduced, phenol red-free</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">3-4% agarose</td>
			<td>Sigma&#160;</td>
			<td>A9414</td>
			<td style="width:444px;">prepare w/v in 1x DPBS, heating to dissolve in a waterbath</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaging of fixed intestines</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td style="width:444px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vaseline</td>
			<td style="width:223px;">purchase at pharmacy</td>
			<td></td>
			<td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lanolin</td>
			<td style="width:223px;">Sigma&#160;</td>
			<td>L7387</td>
			<td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paraffin</td>
			<td style="width:223px;">Surgipath</td>
			<td>39601006</td>
			<td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% glycerol in 1 x PBS</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Focus clear and Mount Clear</td>
			<td>CelExplorer Labs Co.</td>
			<td>&#160;F101-KIT</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Modeling clay</td>
			<td>purchase at art supply store</td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">double stick tape</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cotton applicator swabs</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">plastic molds, 10mm x 10mm x 5 mm)</td>
			<td>Tissue Tek&#160;</td>
			<td>4565</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">slides</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">coverslips</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lab wipe</td>
			<td>Kimberly Clark</td>
			<td>34155</td>
			<td style="width:444px;">lint free delicate task wipe</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Theiler staging chart&#160;</td>
			<td></td>
			<td></td>
			<td style="width:444px;"><a href="http://www.emouseatlas.org/emap/ema/theiler_stages/%20downloads/theiler2.pdf">http://www.emouseatlas.org/emap/ema/theiler_stages/ downloads/theiler2.pdf</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica SP5X confocal microscope&#160;</td>
			<td>Leica</td>
			<td></td>
			<td style="width:444px;">Used to conduct the live imaging&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica DMI 6000 stand&#160;</td>
			<td>Leica</td>
			<td></td>
			<td style="width:444px;">Used to conduct the live imaging&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Aqueous mounting medium (Prolong Gold)</td>
			<td>Molecular Probes&#160;</td>
			<td>P36930</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials for Immunofluorescence staining of fixed, vibratome sectioned intestines</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td style="width:444px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well plate</td>
			<td>Costar</td>
			<td>&#160;3524</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Triton X-100</td>
			<td>Sigma&#160;</td>
			<td>T-8787</td>
			<td style="width:444px;">used to make Permeabilization solution: 0.5% Triton X-100 in 1 x PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Goat serum</td>
			<td></td>
			<td></td>
			<td style="width:444px;">used to make Blocking Solution: 4% Goat serum, 0.1% Tween20 in 1x PBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Tween20&#160;</td>
			<td>Sigma</td>
			<td>&#160;P9416</td>
			<td style="width:444px;"></td>
		</tr>
	</tbody>
</table>

mouse fetal whole intestine culture system for ex vivo manipulation of signaling pathways and three-dimensional live imaging of villus development

태아 소장에서 대부분의 형태 형성 과정은 발달 단계를 통해 변화의 스냅 샷을 제공하는 고정 된 조직의 얇은 섹션에서 유추되고있다. 얇은 시리얼 섹션에서 세 가지 차원 정보로 인해 완벽하게 시리얼 섹션을 재구성 시리얼 섹션을 통해 조직의 올바른 방향을 유지의 어려움의 해석에 도전 할 수 있습니다. 그로 의해 외. 최근 연구 결과, 2011은 한 부위의 현상 융모의 형태 형성을 이해 삼차원 정보의 중요성을 강조. 단독 표지 장 세포의 3 차원 재구성은 장 상피 세포의 대부분은 모두 혀끝과 기저 표면에 문의하는 것이 보여 주었다. 더욱이, 상피의 선 단면에서 액틴 세포 골격의 삼차원 재구성 장내 루멘 연속이며 그 보조 루멘 (S)의 이슈임을 입증ectioning. 그 두 점은, 장 상피 세포에서 interkinetic 핵 마이그레이션 시연과 함께 pseudostratified 상피 세포로 개발하는 장 상피 세포를 정의하고 이전에 한 생각으로 계층화 없습니다. 상피 세 차원을 관찰 할 수있는 능력은이 점을 증명하고 태아 소장에서 상피 형태 형성을 재정에 정액이었다. 다중 광자 이미징 기술 및 3 차원 재구성 소프트웨어의 발전으로, 능력은 급속히 향상되고 장기 개발, 그대로 시각화. 이광자 여기는 고해상도 조직에 덜 파괴적인 침투를 깊게한다. 두 광자 이미징 및 월튼 등.에 전체 태아 마우스 내장의 3 ​​차원 복원은 2012 융모 파생물이의 패턴을 정의하는 데 도움을 주었다. 여기에서는 허용하도록 그 배양 시스템의 융모와 확장 생체 개발을 허용 전체 기관 배양 시스템을 설명창자는 세 가지 차원으로 자신의 개발 기간 동안 이미지를 만들 수 있습니다.

이 시스템은 약리학 적 시약 또는 재조합 단백질과 시그널링의 조작을 가능으로 태아 내장의 전체 이식편의 배양은, 장 개발 좌표 시그널링 분자의 위치, 분포, 및 지속 시간의 분석을 허용한다. (월튼 외로부터 재생도 1a., 2012) 트랜스 웰 배양 시스템 (2)은 가능한 조직에 약물이나 단백질 침지 아가 로스 비드를 배치함으로써, 로컬 신호 원을 확립하게 공기 - 액?...

Watch this Scientific Journal Video about Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development at JoVE.com

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

에 대한 마우스 태아의 전체 소장 문화 시스템<em&gt; 전의 VIVO</em&gt; 신호 전달 경로와 융모 개발의 3 차원 라이브 영상의 조작

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

mouse-fetal-whole-intestine-culture-system-for-ex-vivo-manipulation

Research

JoVE Journal

Biology

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

14.7K 조회수.  University of Michigan. Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine. 

비디오: Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

조기 칙 태아 예 생체내 (새로운 문화)의 문화에 대한 방법

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

연구

생물학

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 ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

전 비켜</em이미징 및 미세 애플 리케이션에 적합> 치킨의 배아 문화 시스템

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

회전 형 바틀 문화 시스템을 사용 로던트 전체 엠브료 문화의 방법

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

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 ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

DNA의 손상 응답 단백질의 2와 3 차원 라이브 셀 이미징

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

무 혈청 전체 배아 배양 시스템에서 마우스 배아 개발

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

마우스 배아의 피부와 Melanoblast 이주의 라이브 영상의 생체 문화

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

<em&gt; 예 생체 내</em&gt; 문화 시스템 갑상선 개발을 연구하는

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

3 차원 히드로 겔의 이미지를 FRET

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...