저자는 Leif Oxburgh와 Derek Adams에게 도움을 주신 Leif Oxburgh와 Stefan Wölfl 및 Ulrike Müller와 Saskia Schmitteckert, Julia Gobbert, Sascha Weyer 및 Viola Mayer에게 도움을 주신 데 대해 감사드립니다. 랩. 이 작품은 개발, 생물 학자의 회사 (CP) 에 의해 지원되었다 .

마우스는 스웨덴의 규정 및 EU 법령 (2010 / 63 / EU)에 따라 유지 관리되었습니다. 모든 절차는 스웨덴 윤리위원회 (C79 / 9, C248 / 11 및 C135 / 14 허가)의 지침에 따라 수행되었습니다. 동물과 관련된 하이델베르그 대학의 절차는 하이델베르그 대학의 Regierungspräsidium Karlsruhe와 Animal Welfare Officers에 의해 승인되었습니다. 1. 시약 및 시약의 준비 참고 : 오염을 최소화하려면 층류 후드를 사용하십시오. <ol><li> 해부 당일에, 무균 인산 완충 식염수 (PBS) 중의 1 : 5의 몰비로 항 - 인간 Fc 염소 항체와 혼합함으로써 인간 Fc 단독 또는 인간 Fc에 융합 된 재조합 키메라 에프 린 단백질을 사전 - 클러스터링한다. 37 ° C에서 1 시간 동안 품어 둡니다. 5 . </li><li> Dulbecco &#39;s Modified Eagle Medium : Nutrient를 보충하여 배양 배지를 준비하십시오.1 % 페니실린 / 스트렙토 마이신 (v / v), 1 % 글루타민 (v / v), 1 % 인슐린 - 트랜스페린 - 셀레늄 (v / v), 50 μg / ml 인간 홀로 코 - 트랜스페린 및 1.5㎍ / mL 암포 테리 신이 포함된다. 참고 : 16 ML 배아의 10 ML 충분합니다. </li><li> 배양액에 최종 농도 20 μg / mL의 클러스터 된 재조합 에프린 용액을 첨가합니다. 클러스터 된 인간 Fc를 대조군으로 사용하십시오. </li><li> 무균, 코팅되지 않은 평평한 바닥 폴리스티렌 4 잘 접시의 각 우물에 문화 매체 500 μL를 추가하여 문화 플레이트를 준비합니다. </li><li> 무균 포셉을 사용하여 신중하게 매체 상단에 구멍 당 5 μm의 기공 크기와 하나의 폴리 카보 네이트 멤브레인 필터를 놓으십시오. 인큐베이터 (37 ° C / 5 % CO 2 )에 준비 접시를 전송하십시오. 필터가 윗면이 마를 때까지 뜨고 플로트가 발생하는지 확인하십시오. 참고 : 두 개의 신장 혈관에 대해 하나의 필터로 충분합니다. </li><li> 면도날을 사용하여 대략 30 피펫 팁 (볼륨 : 100 μL) approximately 끝에서 1mm가 더 넓게 열립니다. 팁을 다시 피펫 - 팁 랙에 넣으십시오. </li></ol> 2. E11.5에서 Metanephric Rudiments의 해부 <ol><li> 자궁 경부 탈구 (또는 지역 윤리위원회의 승인을받은 방법을 사용)하여 E11.5에서 임신 임신 한 마우스를 희생합니다. </li><li> Brown 외 6 에 설명 된대로 자궁과 배아를 제거합니다. 이 시점에서 해부 현미경으로 작업하십시오. </li><li> 5 번 시계 제조기 겸자를 사용하여 앞다리 바로 아래에서 배아를 잘라냅니다. 각 배아에 신선한 페트리 접시를 사용하십시오. 배아의 꼬리와 다리를 제거하십시오. 이렇게하려면 5 번 시계 제조기 집게 한 쌍으로 제거 할 조직을 잡고 두 번째 쌍의 끝을 사용하여 자릅니다. </li><li> 복부 기관의 질량을 제거하려면, 꼬리의 주둥이 방향으로 배아의 몸 벽을 옆으로 열려면 한 쌍의 포셉의 팁을 사용하십시오. <ol><li> 외형 적으로 절단, t와 평행하게그는 지느러미 대동맥. 오리 엔테이션을 위해 혈액으로 채워지기 때문에 눈에 보이는 대동맥을 사용하십시오. 조심스럽게 폐쇄 포셉의 팁을 사용하여 등쪽 부분에서 신체 벽의 복부 부분을 분리하고 배아를 뒤집습니다. 앞면과 마찬가지로 몸통 벽을 옆으로 대동맥 - 꼬리 방향으로 절단하고 배부 대동맥을 사용하여 방향을 잡습니다. </li></ol></li><li> 복부 몸의 벽이 지느러미 몸체에서 분리되면, 한 쌍의 포셉으로 잡아서 신중하게 잡아 당겨 기관 덩어리와 함께 제거하십시오. 참고 : metanephric anlagen은 일반적으로 등쪽 몸 벽에 붙어있어. 그들은 타원형이며, 뒷다리 수준에 위치한 ~ 200 μm 길이의 구조물입니다. </li><li> 복부 쪽이 위로 향하게 한 쌍의 포 셉를 사용하여 등쪽 몸통 벽을 지킨다. 포셉의 다른 한 켤레를 사용하여, 그것의 주둥이 끝에서 등쪽 대동맥을 잡고 조심스럽게 몸의 벽에서 등 대동맥을 껍질. 참고 : 두 metanephric 신장은 일반적으로 붙어있어등 대동맥에 붙어있다. </li><li> 한 쌍의 포셉을 사용하여 신장 anlagen에 rostrally 잘라 대동맥을 제거하십시오. 그런 다음 신중하게 포 셉의 팁을 사용하여 두 신장 anlagen 분리하십시오. </li><li> 조심스럽게 포셉의 팁을 사용하여 신장 anlagen에서 원치 않는 조직을 껍질. mesenchyme을 손상시키지 않도록주의하십시오. 손상은 추가 분석에서 explant의 성장 및 배제를 초래할 수 있습니다. </li><li> 4 잘 접시에 폴리 카보 네이트 멤브레인 필터에 PBS 10 μL에 microliter 피펫과 와이드 오픈 피펫 팁을 사용하여 신장 anlagen을 별도로 전송합니다. 참고 : 표면 장력으로 인해 체외 이식편은 얇은 매질로 덮여 있습니다. 하나의 필터로 두 개의 신장 혈관이 충분합니다. <ol><li> 두 개의 신장 anlagen 각 필터의 ​​각 절반의 중심에 놓습니다. 37 ° C / 5 % CO 2 에서 배양기로 플레이트를 다시 옮긴다. metanephric rud 때까지 인큐베이터에 접시를 남겨주세요다음 배아에서 iments은 필터에 배치 할 준비가되었습니다. </li></ol></li><li> 각 배아에 대해 2.3-2.9.1 단계를 반복합니다. 참고 : 일부 연습으로 해부 절차는 배아당 약 5 분 걸립니다. </li><li> 37 ° C / 5 % CO 2 에서 3 일 동안 신장 anlagen을 품어. </li></ol> 3. 고정 및 염색을위한 시약의 준비 <ol><li> 칼슘과 마그네슘 (w / v)이없는 PBS에서 4 % 파라 포름 알데히드 (PFA)를 준비하려면 PFA를 60 ℃에서 PBS에 녹여서 지속적으로 저어줍니다. 실온으로 식힌 다음 페이퍼 필터를 사용하여 남아있는 입자를 제거합니다. 분주하고 4 ° C에서 보관하여 즉시 사용하거나 장기간 보관할 수 있습니다. 주의 : PFA는 유독합니다. 지역 안전 표준에 명시된 개인 보호 장비를 착용하고 흄 후드에서 작업하십시오. 시설 지침에 따라 지정된 폐기물 용기를 사용하여 폐기물을 버립니다. </li><li> permeabilization solution을 준비하기 위해 희석 된 Triton X-1000.3 % (v / v)가되도록 칼슘과 마그네슘이없는 PBS에서 처리 하였다. </li><li> 차단 용액을 준비하기 위해 5 % (v / v) 염소 혈청과 0.1 % Triton X-100을 칼슘과 마그네슘없이 PBS에 첨가한다. 나트륨 아자 이드를 0.02 % (w / v)의 최종 농도로 첨가한다. 4 ° C에서 보관하십시오. 주의 : 나트륨 아자 이드는 독성이 있습니다. 지역 안전 및 환경 보호 규정에 따라 나트륨 아자 이드를 취급하십시오. 참고 : 염색을 위해 항체를 사용하는 경우 2 차 항체가 유도 된 종에서 5 % 혈청을 사용하여 차단 용액을 만듭니다. </li><li> biotinylated Dolichorus biflorus agglutinin을 1 : 200으로 블로킹 용액으로 희석한다. Alexa488 - conjugated streptavidin을 1 : 200으로 블로킹 용액으로 희석하십시오. </li><li> 즉시 사용할 수있는 배지 (물질 표 참조)를 만들기 위해 0.1M Tris-HCl (pH 8.5)에 녹인 25 % (w / v) 글리세롤에 0.1g의 수용성 폴리 비닐 알코올 계 점착제 / mL를 넣는다. . 연속 교반하에 50 ° C로 1 시간 가열하고 식힌 다음 1을 사용하여 pH를 8.0-8.5로 조정한다.M NaOH. NaOH 농도를 높이 지 마십시오. 0.02 % (w / v)의 최종 농도로 100 μg / mL의 N- 프로필 게 네이트와 티 메로 살을 첨가한다. 실온에서 30 분 동안 교반한다. 주의 : Thimerosal은 독성이 있습니다. 해당 지역의 안전 및 환경 보호 규정에 따라 취급하십시오. <ol><li> 50 ML 튜브에 삽입 매체를 채우고, 로터를 균형을, 그리고 3,200 XG 및 10 분 실온에서 원심 분리기. 15 mL 튜브에 투명한 용액을 따라 내고 펠렛을 버린다. 분주하고 -20 ℃에서 몇 주 동안 보관하십시오. 해동되면 용액을 4 ° C에 유지한다. 사용 직전 ~ 30 ° C로 예열하십시오. </li></ol></li><li> explants와 필터가 장착 될만큼 많은 유리 슬라이드를 준비합니다. 이를 위해 인스턴트 접착제를 사용하여 유리 슬라이드 양쪽에 18 x 18 mm 크기의 작은 커버 슬립 2 개를 붙이고 필터 사이에 15 mm의 공간을 남겨 둡니다. </li></ol> 4. 고정 및 염색 <ol><li> 배양 기간 3배양기에서 플레이트를 제거하고 조심스럽게 micropipette를 사용하여 각 우물에서 문화 매체를 대기음. 필터를 만지지 말고 팁 입구를 우물 벽쪽으로 유지하십시오. </li><li> 각 우물에 4 % PFA / PBS 500 μL를 추가합니다. 필터는 정착액 위에 뜨게됩니다. 마이크로 피펫을 사용하여 PFA 고정액을 조심스럽게 첨가하여 필터를 잠수시킵니다. 실온에서 30 분 동안 품어 낸다. 참고 : 다음 단계를 수행 할 때 필터에서 외과 체를 씻어 내지 않도록주의해야합니다. 우물 벽을 향한 피펫 팁의 개방을 유지하십시오. </li><li> PFA를 제거하고 500 μL의 PBS로 두 번 헹구십시오. 각 우물에 permeabilization 솔루션 500 μL를 추가하고 1 시간 동안 실온에서 품어. </li><li> PBS 500 μL로 두 번 씻어서 각 잘 차단 솔루션의 500 μL를 추가합니다. 실온에서 1 시간 동안 인큐베이션한다. </li><li> 차단 용액을250 μL의 biotinylated Dolichorus biflorus agglutinin은 1 : 200으로 블로킹 용액으로 희석하고 4 ° C에서 24 시간 동안 배양한다. </li><li> Dolichorus biflorus agglutinin 솔루션을 제거하고 잘 PBS 당 500μL로 씻어. </li><li> Alexa488 - conjugated streptavidin 250 μL를 1 : 200 희석 용액에 첨가하고 4 ° C에서 24 시간 동안 배양하십시오. 또는 실온에서 2 시간 동안 배양하십시오. 참고 : 4 ° C에서 배양하면 더 나은 신호 대 잡음비가 달성됩니다. </li><li> 1 웰당 PBS 500 μL로 두 번 씻어. 집게를 사용하여 explants이 위로 향하게 준비 슬라이드에 필터를 전송합니다. 임베딩 매체 ~ 50-100 μL로 탑재하십시오. 60mm 길이의 1.5 번 커버 슬립으로 덮으십시오. 매질 용액을 실온에서 2 시간 동안 응고시킨다. 이미지 준비가 끝날 때까지 4 ° C에서 포일로 싸인 슬라이드를 이미지화하거나 보관하십시오. </li><li> 와이드 필드 epifluorescence 현미경 7 &lt;/ sup&gt;를 Hg- 램프에 연결하고 청색 여기 (여기 대역 통과, 460 - 495 nm, 이색 거울, 505 nm, 방출 대역, 510 - 550 nm) 및 20X Plan-Apochromat 렌즈 용 거울 장치를 사용하여 0.75 구멍. </li></ol>

<ol><li>Saxén, L. <a target="_blank" href="http://admin.cambridge.org/fk/academic/subjects/life-sciences/cell-biology-and-developmental-biology/organogenesis-kidney">Organogenesis of the kidney. Developmental and Cell Biology Series</a>. 19, Cambridge University Press. (1987).</li>
<li>Lindström, N. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrated+%CE%B2-catenin%2C+BMP%2C+PTEN%2C+and+Notch+signalling+patterns+the+nephron%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Integrated β-catenin, BMP, PTEN, and Notch signalling patterns the nephron.</a> eLife. 4, e04000(2015).</li>
<li>Peuckert, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multimodal+Eph%2FEphrin+signaling+controls+several+phases+of+urogenital+development%5BTitle%5D)+AND+%22Kidney+Int%22%5BJournal%5D)">Multimodal Eph/Ephrin signaling controls several phases of urogenital development.</a> Kidney Int. 90 (2), 373-388 (2016).</li>
<li>Pasquale, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Eph+receptor+signalling+casts+a+wide+net+on+cell+behaviour%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">Eph receptor signalling casts a wide net on cell behaviour.</a> Nat Rev Mol Cell Biol. 6 (6), 462-475 (2005).</li>
<li>Bonanomi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ret+Is+a+Multifunctional+Coreceptor+that+Integrates+Diffusible-+and+Contact-Axon+Guidance+Signals%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Ret Is a Multifunctional Coreceptor that Integrates Diffusible- and Contact-Axon Guidance Signals.</a> Cell. 148 (2), 568-582 (2012).</li>
<li>Brown, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+Culture+of+Cells+from+the+Nephrogenic+Zone+of+the+Embryonic+Mouse+Kidney%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney.</a> J Vis Exp. (50), e2555(2011).</li>
<li>Olympus Support. , Available from:
 <a target="_blank" href="http://www.olympusamerica.com/cpg_section/cpg_archived_product_details.asp?id=817">http://www.olympusamerica.com/cpg_section/cpg_archived_product_details.asp?id=817</a> (2016).</li>
<li>Grobstein, C. <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1002/jez.1401300207/abstract">Inductive interaction in the development of the mouse metanephros.</a> J Exp Zool. 130, 319-340 (1955).</li>
<li>Saxén, L., Toivonen, S. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/nlmcatalog/38101">Primary Embryonic Induction</a>. , Academic Press. London. (1962).</li>
<li>Saxén, L., Koskimies, O., Lahti, A., Miettinen, H., Rapola, J., Wartiovaara, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiation+of+kidney+mesenchyme+in+an+experimental+model+system%5BTitle%5D)+AND+%22Adv+Morphog%22%5BJournal%5D)">Differentiation of kidney mesenchyme in an experimental model system.</a> Adv Morphog. 7, 251-293 (1968).</li>
<li>Dudley, A. T., Godin, R. E., Robertson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interaction+between+FGF+and+BMP+signaling+pathways+regulates+development+of+metanephric+mesenchyme%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">Interaction between FGF and BMP signaling pathways regulates development of metanephric mesenchyme.</a> Genes Dev. 13, 1601-1613 (1999).</li>
<li>Perälä, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sema4C-Plexin+B2+signalling+modulates+ureteric+branching+in+developing+kidney%5BTitle%5D)+AND+%22Differentiation%22%5BJournal%5D)">Sema4C-Plexin B2 signalling modulates ureteric branching in developing kidney.</a> Differentiation. 81 (2), 81-91 (2011).</li>
<li>Thesleff, I., Ekblom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+transferrin+in+branching+morphogenesis%2C+growth+and+differentiation+of+the+embryonic+kidney%5BTitle%5D)+AND+%22J+Embryol+exp+Morph%22%5BJournal%5D)">Role of transferrin in branching morphogenesis, growth and differentiation of the embryonic kidney.</a> J Embryol exp Morph. 82, 147-161 (1984).</li>
<li>Watanabe, T., Costantini, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+analysis+of+ureteric+bud+branching+morphogenesis%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Real-time analysis of ureteric bud branching morphogenesis.</a> Dev Biol. 271, 98-108 (2004).</li>
<li>Sebinger, D. D. R., Unbekandt, M., Ganeva, V. V., Ofenbauer, A., Werner, C., Davies, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Novel%2C+Low-Volume+Method+for+Organ+Culture+of+Embryonic+Kidneys+That+Allows+Development+of+Cortico-Medullary+Anatomical+Organization%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">A Novel, Low-Volume Method for Organ Culture of Embryonic Kidneys That Allows Development of Cortico-Medullary Anatomical Organization.</a> PLoS One. 5 (5), e10550(2010).</li>
<li>Ekblom, P., Miettinen, A., Virtanen, I., Wahlström, T., Dawnay, A., Saxén, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+segregation+of+the+metanephric+nephron%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">In vitro segregation of the metanephric nephron.</a> Dev Biol. 84 (1), 88-95 (1981).</li>
<li>Davies, J. A., Unbekandt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((siRNA-mediated+RNA+interference+in+embryonic+kidney+organ+culture%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">siRNA-mediated RNA interference in embryonic kidney organ culture.</a> Methods Mol Biol. 886, 295-303 (2012).</li>
<li>Saxén, L., Lehtonen, E. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/3328727">Embryonic kidney in organ culture.</a> Differentiation. 36 (1), 2-11 (1987).</li>
<li>Bard, J. B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+development+of+the+mouse+kidney+embryogenesis+writ+small%5BTitle%5D)+AND+%22Curr+Opin+Genet+Dev%22%5BJournal%5D)">The development of the mouse kidney embryogenesis writ small.</a> Curr Opin Genet Dev. 2, 589-595 (1992).</li>
<li>Davies, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+method+for+cold+storage+and+transport+of+viable+embryonic+kidney+rudiments%5BTitle%5D)+AND+%22Kidney+Int%22%5BJournal%5D)">A method for cold storage and transport of viable embryonic kidney rudiments.</a> Kidney Int. 70 (11), 2031-2034 (2006).</li>
<li>Batourina, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distal+ureter+morphogenesis+depends+on+epithelial+cell+remodeling+mediated+by+vitamin+A+and+Ret%5BTitle%5D)+AND+%22Nat+Genet%22%5BJournal%5D)">Distal ureter morphogenesis depends on epithelial cell remodeling mediated by vitamin A and Ret.</a> Nat Genet. 32 (1), 109-115 (2002).</li>
</ol>

저자는 공개 할 것이 없습니다.

이 원고는 마우스 배아에서 발생하는 metanephric anlagen을 분리하고 장기 기초를 배양하는 방법을 설명합니다. 이 방법은 Grobstein 8 과 Saxén 9 , 10 에 의해 개발 된 표준 기법이며 많은 다른 사람들에 의해 수정되고 수정되었습니다 11 , 12 . 이 방법의 성공은 주로 절개의 지속 기간과 장기간의 절?...

The mammalian kidney is derived from two primordial structures with mesodermal origin: the tubular epithelial ureteric bud and the metanephric mesenchyme. During nephrogenesis, the ureteric bud invades the metanephric mesenchyme and branches to form the collecting system. The metanephric mesenchyme gives rise to the epithelial elements of the nephrons. These processes occur in a precisely timed and spatially coordinated manner and are initiated by reciprocal inductive mechanisms. Both tissue components communicate and affect the other's cell morphogenesis. 
In the 1920s, it was Boyden who performed the in vivo obstruction of the mesonephric duct in chicken, providing the first indication of inductive interactions as separated nephric blastema fail to differentiate1. At about the same time, the first successful attempts to culture chicken nephric rudiments in a hanging drop were published. Subsequently, the organ culture was developed to study tissue interactions in mammalian organogenesis. In the 1950s, Grobstein developed a technique in which metanephric rudiments could be cultured on a filter. This technique was modified by Sax&#233;n, who placed the filter on a Trowell-type screen in a culture dish1. Over the years, many modifications and applications for organ culture have emerged. The method described here is based on Sax&#233;n's technique but is simplified, as the filters float free on the medium and the diameter of the culture well only slightly exceeds the diameter of the filter, limiting unwanted movement of the filter.
Whole-organ culture is a classical, cheap, and simple but powerful tool to investigate cellular processes and intercellular communication during organogenesis. Organ culture allows for treatment with biological agents, such as growth factors, antibodies, antisense oligonucleotides, viruses, and peptides, as well as with pharmaceutical compounds and other chemicals. Also, gene function may be studied using explants derived from genetically modified mice or using inducible gene inactivation technology, such as the Cre-loxP system. This allows for the study of genetic mutations that cause embryonic lethality prior to the development of the kidney. Organ culture can also be combined with fluorescent tagging for gene function or lineage tracing and modern imaging techniques, which enable real-time monitoring of cell behavior2.
In the specific example provided here, the effect of EphrinB2-activated Eph-receptor signaling on the branching morphology of the ureteric bud was investigated. The morphology of the EphA4/EphB2 double-knockout mice suggested several severe defects in kidney development, which were detectable as early as embryonic day 11 (E11) and involved the ureteric bud, the ureter, and the common nephric duct3. Signaling via Eph receptors requires the clustering of the ligand-receptor dimer4. To over-activate Eph signaling, the kidney rudiments from E11.5 mouse embryos were cultured in the presence of clustered recombinant EphrinB2-Fc. EphrinB2 is a known ligand for the EphA4 receptor, which is expressed in the ureteric bud tips3.

<table><tbody><tr><td>DMEM/F-12</td><td>Thermo Fisher Scientific</td><td>21331020</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Thermo Fisher Scientific</td><td>15140148</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>Thermo Fisher Scientific</td><td>35050061</td><td></td></tr><tr><td>DPBS, calcium, magnesium</td><td>Thermo Fisher Scientific</td><td>14040117</td><td>use for dissection</td></tr><tr><td>holo-Transferrin human</td><td>Sigma-Aldrich</td><td>T0665</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium (ITS -G) (100x)</td><td>Thermo Fisher Scientific</td><td>41400045</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Amphotericin B solution</td><td>Sigma-Aldrich</td><td>A2942</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S8032</td><td></td></tr><tr><td>Thimerosal</td><td>Sigma-Aldrich</td><td>T5125</td><td></td></tr><tr><td>Propyl gallate</td><td>Sigma-Aldrich</td><td>2370</td><td></td></tr><tr><td>Mowiol 4-88</td><td>Sigma-Aldrich</td><td>81381</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Biotinylated Dolichorus Biflorus Agglutinin</td><td>Vector Laboratories</td><td>B-1035</td><td></td></tr><tr><td>Alexa488 conjugated Streptavidin</td><td>Jackson Immuno Research</td><td>016-540-084</td><td></td></tr><tr><td>Recombinant Mouse Ephrin-B2 Fc Chimera Protein, CF</td><td>R&amp;amp;D Systems</td><td>496-EB</td><td></td></tr><tr><td>Recombinant Human IgG1 Fc, CF</td><td>R&amp;amp;D Systems</td><td>110-HG-100</td><td></td></tr><tr><td>Goat Anti-Human IgG Fc Antibody</td><td>R&amp;amp;D Systems</td><td>G-102-C</td><td></td></tr><tr><td>Phosphate buffered saline tablets</td><td>Sigma-Aldrich</td><td>P4417</td><td>use for fixation and immunostaining</td></tr><tr><td>Dumont #5, biologie&lt;br/&gt; tips, INOX, 11 cm</td><td>agnthos.se</td><td>0208-5-PS</td><td>2 pairs of forceps are needed</td></tr><tr><td>Iris scissors, straight, 12 cm</td><td>agnthos.se</td><td>03-320-120</td><td></td></tr><tr><td>Dressing Forceps,&lt;br/&gt; straight, delicate, 13 cm</td><td>agnthos.se</td><td>08-032-130</td><td></td></tr><tr><td>Petri dishes Nunclo Delta treated</td><td>Thermo Fisher Scientific</td><td>150679</td><td></td></tr><tr><td>TMTP01300 Isopore Membrane Filter, polycarbonate, Hydrophilic, 5.0 &amp;micro;m, 13 mm, white, plain</td><td>MerckMillipore</td><td>TMTP01300</td><td></td></tr><tr><td>Nunclon Multidishes&lt;br/&gt; 4 wells, flat bottom</td><td>Sigma-Aldrich</td><td>D6789-1CS</td><td></td></tr><tr><td>Microscope cover glass 24 x 50 mm thickn. No.1.5H 0.17+/-0.005 mm</td><td>nordicbiolabs</td><td>107222</td><td></td></tr><tr><td>Cover glasses No.1.5, 18 mm x 18 mm</td><td>nordicbiolabs</td><td>102032</td><td></td></tr><tr><td>Slides ~76 x 26 x 1, 1/2-w. ground plain</td><td>nordicbiolabs</td><td>1030418</td><td></td></tr><tr><td>VWR Razor Blades</td><td>VWR</td><td>55411-055</td><td></td></tr><tr><td>50 mL centrifuge tubes</td><td>Sigma-Aldrich</td><td>CLS430828</td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td>Sigma-Aldrich</td><td>CLS430055</td><td></td></tr><tr><td>Whatman prepleated qualitative filter paper, Grade 113V, creped</td><td>Sigma-Aldrich</td><td>WHA1213125</td><td></td></tr><tr><td>Fixed stage research mircoscope</td><td>Olympus</td><td>BX61WI</td><td></td></tr><tr><td>Black 6 inbred mice, male, C57BL/6NTac</td><td>Taconic</td><td>B6-M</td><td></td></tr><tr><td>Black 6 inbred mice,female, C57BL/6NTac</td><td>Taconic</td><td>B6-F</td><td></td></tr><tr><td>Greenough Stereo Microscope</td><td>Leica</td><td>Leica S6 E</td><td></td></tr></tbody></table>

dissection and culture of mouse embryonic kidney

이 프로토콜의 목표는 마우스 metanephric 기초의 해부, 격리 및 문화에 대한 방법을 설명하는 것입니다. 포유류 신장이 발달하는 동안 두 개의 전구 세포 조직, 즉 ureteric bud와 metanephric mesenchyme은 세포 기작을 전달하고 상호 작용하여 결국 포집 시스템과 신장의 네프론을 형성합니다. 포유류 배아가 자궁 내에서 자라며 관찰자가 접근하기 어렵 기 때문에 장기 배양이 개발되었습니다. 이 방법을 사용하면 신장 조직 형성 과정에서 상피 간엽 상호 작용과 세포 행동을 연구 할 수 있습니다. 또한 선천성 신장 및 비뇨 생식기 기형의 기원을 조사 할 수 있습니다. 조심스럽게 해부 된 후, metanephric 기초 물질은 배양 배지에 떠있는 필터로 옮겨져 며칠 동안 세포 배양기에 보관 될 수 있습니다. 그러나 조건은 다음과 같습니다.인공이며 조직의 신진 대사에 영향을 줄 수 있습니다. 또한, 시험 물질의 침투는 체외 이식편에 존재하는 세포 외 기질 및 기저막으로 인해 제한 될 수있다. 기관 문화의 한 주요 이점은 실험자가 기관에 직접 접근 할 수 있다는 것입니다. 이 기술은 저렴하고 간단하며 생물학적 활성 물질의 첨가, 유전 변이의 연구, 진보 된 영상 기술의 적용과 같은 많은 수정을 가능하게합니다.

Metanephric kidn anlagen은 E11.5에서 임신 한 Black-6 근친 교배 마우스에서 추출하여 배양 하였다. 3 일 후, ureteric bud는 5 번까지 분지되었고, 그 결과 초기 T 자 형태의 ureteric bud가 분출되었다. 각 체외 이식편을 촬영하고 분지 생성량을 결정하고 분지 당 종말점 수를 계산하기 위해 분절 수와 종점을 정량화했습니다 ( 그림 1 ). ImageJ ( 제 4 세대), 제 4 세대는 처...

Watch this Scientific Journal Video about Dissection and Culture of Mouse Embryonic Kidney at JoVE.com

Dissection and Culture of Mouse Embryonic Kidney

이 프로토콜은 마우스 배아에서 metanephric의 기초를 분리하고 배양하는 방법을 설명합니다.

마우스 배아 신장의 해부 및 배양

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

dissection-and-culture-of-mouse-embryonic-kidney

Research

JoVE Journal

Developmental Biology

12.0K 조회수.  Uppsala University.  이 프로토콜은 마우스 배아에서 metanephric의 기초를 분리하고 배양하는 방법을 설명합니다. 

비디오: Dissection and Culture of Mouse Embryonic Kidney

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.
Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.

A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.

마우스 배아 전체 문화 신경 튜브 폐쇄

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010).  However, the study of mouse ...

연구

신경과학

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

오르간 문화와 마우스 Wolffian 덕트의 전체 마운트 면역 형광 염색법

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

발생학

Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy Imaging

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental processes and modelling kidney disease. However, the models are limited by a lack of vascularization and functionality. To address this, an improved protocol for the method of xenografting cells and tissues to the chorioallantoic membrane (CAM) of an avian embryo to gain vascularization and restoration of blood flow was developed. The grafts are overlaid with custom-made minireservoirs that fix the samples to the CAM and supply them with culture medium that protects the grafts from drying. The improved culture method allows xenografts to grow for up to 9 days. The manuscript also describes how to provide optimal conditions for long-term confocal imaging of renal organoids and organotypic cultures using the previously published Fixed Z-Direction (FiZD) method. This method gently compresses an embryonic organ or organoid between a glass coverslip and membrane in a large amount of medium and provides excellent conditions for imaging for up to 12 days. Together, these methods allow vascularization and blood flow to renal organoids and organotypic kidney cultures with improved confocal imaging. The methods described here are highly beneficial for studying fundamental and applied functions of kidneys ex vivo. Both methods are applicable to various types of tissues and organoids.

This work describes two methods for studying organ development, an improved xenotransplantation setup on chorioallantoic membrane (CAM) from avian embryos that allows for vascularization of cultured embryonic organs and organoids and a novel fixed z-direction organ culture method with modified experimental conditions that allows for high-resolution time-lapse confocal imaging.

혈관 을 위한 신장 오르가노이드 및 Organotypic 문화의 최적화, 확장된 발달 및 향상한 현미경 검사법 화상 진찰

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

마우스 배아의 폐 문화, 분기의 분자 메커니즘을 평가하는 시스템

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

생물학

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

서 주 신장 조직 세그먼트와 소설 비 계 없는 건설 기술 관

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

생체공학

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

배아 마우스 신장의 Nephrogenic 지대의 세포의 분리 및 문화

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

절연 및 마우스 배아 신경 줄기 세포의 문화

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

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 ...

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of pathways involved in parietal epithelial cell activation could therefore be a tool to attenuate the progression of glomerular diseases. This article describes a method to culture and analyze parietal epithelial cell outgrowth of encapsulated glomeruli isolated from mouse kidney. After dissecting isolated mouse kidneys, the tissue is minced, and glomeruli are isolated by sieving. Encapsulated glomeruli are collected, and single glomeruli are cultured for 6 days to obtain glomerular outgrowth of parietal epithelial cells. During this period, parietal epithelial cell proliferation and migration can be analyzed by determining the cell number or the surface area of outgrowing cells. This assay can therefore be used as a tool to study the effects of an altered gene expression in transgenic- or knockout-mice or the effects of culture conditions on parietal epithelial cell growth characteristics and signaling. Using this method, important pathways involved in the process of parietal epithelial cell activation and consequently in glomerulosclerosis can be studied.

This article describes a method for culturing and analyzing glomerular parietal epithelial cell outgrowths of encapsulated glomeruli isolated from mouse kidney. This method can be used to study pathways involved in parietal epithelial cell proliferation and migration.

정수리 상피 세포 활성화에 관여하는 통로를 분석하는 전 비보 분석으로 구체 아웃스승

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

1차 마우스 망막 색소 상피 세포의 효율적인 해부 및 배양

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...