The authors do not have any acknowledgements.

설명하는 모든 절차는 지역 기관 동물 관리 및 사용위원회의 사전 승인을 포함한 척추 동물의 사용에 대한 지침과 규정에 따라 수행해야합니다. 해부 악기와 문화 미디어 1. 준비 <ol><li> 예비 세탁 모든 악기는 3 %의 과산화수소와 20 분 동안 121 ℃에서 다음 오토 클레이브로 절개하여 사용할 수있다. 후드에서 진공 흡입으로 0.22 μm의 기공 크기의 필터를 사용하여 항생제와 항진균제 솔루션 BGJb 미디어를 필터링합니다. </li></ol> 문화 장비 2. 준비 <ol><li> 70 % 알코올 스프레이, 작은 삼각형의 구개 기관을 지원하는 폴리 카보네이트 멤브레인 필터를 잘라하고 건조 할 수 있습니다. 기관 배양 접시 위에 깨끗하고 살균 등변 삼각형 모양의 와이어 그리드 (20mm)를 배치하여 센터 - 글쎄 장기 배양 접시 (60x15 mm)를 준비합니다. 그 다음 B의 1 ㎖로 잘 채우기GJB 미디어 문화 미디어. 참고 : 용지가 입맛을 잠수없이 그리드의 수준에 도달하기에 충분해야한다. 실험에 .Depending 그리드의 상단에있는 멤브레인 필터, 아래 반짝이는면, (그리드 당 3 ~ 4 막)을 배치, 치료 (단백질, 항체 또는 억제제) 미디어에 추가 할 수 있습니다 </li></ol> 3. 마우스 배아 수집 및 문화에 대한 준비 <ol><li> 야생형 마우스 배아를 얻기 위해, 사용은 13.5 임신 한 CD-1 마우스 유전 적 배경의 피로를 시간이 초과되었습니다. 70 % 에탄올로 해부 영역을 청소합니다. 참고 : 마우스의 다른 균주를 사용할 수 있습니다. 때문에 우리는 더 큰 쓰레기 수의 CD-1 마우스를 선호합니다. </li><li> 승인 된 프로토콜을 사용하여 죽음을 보장하기 위해 자궁 경부 전위 다음에 5 %의 이소 플루 란 E13.5 DPC (일 성교를 게시)에서 임신 한 쥐를 안락사. </li><li> 악기에 복부 머리 스틱을 피하기 위해 복부 복부 표면에 70 % 에탄올 스프레이. 그리고 복부 복강을 엽니 다날카로운 무딘 운영 가위 마이크로 해부 집게를 사용하여 자궁을 찾습니다. 집게를 사용하여, 전체 자궁을 들어 올려 자궁 몸을 절단 및 광 운영 가위로 자궁 뿔의 끝을 몸에서 분리. </li><li> (관리 및 실험 동물의 사용을위한 프로토콜 가이드에 따르면) 얼음 위에 깨끗한 종이 타월에 자궁을 놓습니다. 오염을 방지하기 위해 다른 깨끗한 종이 타월로 자궁을 커버. 사용하기 전에 추가로 70 % 에탄올을 사용하여 스프레이기구를 살균. 자궁과 태아는 얼음에 실험실로 운반 할 수있다. </li><li> 오픈 층류 후드에서 신중하게 날카로운 무딘 운영 가위 마이크로 해부 집게를 사용하여 난황에 작은 구멍을합니다. 개방 폭 넓은 확인하고 부드럽게 난황에서 배아를 외면 화하다하기 위해 밀어 넣습니다. </li><li> 차가운 인산 완충 생리 식염수 1X (PBS)의 pH 7.4와 페트리 접시에 즉시 배아를 전송합니다. 적절한 단계, 예를 보장하기 위해몸체, 머리의 크기와 형태에 대한 사지 해부 현미경 배아 아민. 참고 : 개발의 오른쪽 단계에 있지 배아 실험에 사용되지 않습니다. </li></ol><img alt="그림 2" src="/files/ftp_upload/53063/53063fig2.jpg" /> 그림 2. 사지 버드 형태학은. 앞과 뒷다리는 숫자 사이의 웨빙 들여 쓰기의 정도를 검사한다. E13.5에서, 모든 자리 응축은 숫자 사이의 웨빙과 말초 들여 쓰기가 매우 뚜렷한 표시로, 눈에 띄는. 뒷 자리는 반 하루 뒤에 있습니다 (12) 상단 행 :. 앞다리, 낮은 행 : 뒷다리. 4. 미각 해부 <ol><li> 해부 현미경 PBS와 페트리 접시에 한 번에 하나의 배아를 놓습니다. 사용 작은 해부 가위와 핀셋은 태아의 몸에서 머리를 분리합니다. </li><li> 조심스럽게 집게와 눈높이 위의 마우스 두개골을 들고 (피상악 프로세스) 립 라인의 양측에 두 개의 절개를 만든다. 그리고 하악과 혀를 제거합니다. 눈 높이에서 절단하여 상악 프로세스 영역을 분리하고, 뇌, 눈, 코 중격 및 인접 조직을 제거합니다. </li></ol><img alt="그림 3" src="/files/ftp_upload/53063/53063fig3.jpg" /> 그림 3. 구개 조직이 입천장 선반이 상승했다 13.5 배아에서 수집하지만 연락을하지. 구개 선반이 정적 모델에서 72 시간 동안 공기 미디어 인터페이스에서 필터에 장기 문화에 넣었다. (A) 수준 E13.5 배아에 해부. (B) 블루 스타 해부 동안 집게로 조직을 유지하기 위해 지역의 경계. 사다리꼴 필터 멤브레인에 배치 해부 구개 선반 (C)보기. 배양 접시의 (D) 측면 뷰를 배치 한 후 해부 입맛과 문화 미디어의 추가. <ol start="3"><li> 후방에 전방에서 동양을 돕기 위해 첨부 된 기본 미각을 유지, 구개 구강 측면을 놓고 비중격과 주위 조직을 제거합니다. 숟가락 주걱을 사용하여 매우 신중하게 배양 접시에 그리드에 준비된 필터에 (코 쪽 아래) 구개 선반을 전송합니다. 주 : 적절한 공기 미디어 인터페이스를 허용하는 미디어의 볼륨을 조절합니다. </li><li> 스테레오 현미경, 기본 미각을 해부하고 서로 가까이 구개 선반을 배치 핀셋을 사용합니다. 그들이 접촉에 배치되지 않은 경우 구개 선반 융합되지 않습니다. 입천장의 앞쪽 부분이있는 삼각형의 멤브레인 필터를 잘라. 사다리꼴 모양은 미각의 앞쪽 부분을 국산화에 도움이됩니다. </li><li> 배양 구개 가구 단독으로 또는 2 종 BGJb 배양 배지를 사용하여 동일한 기관 배양 접시에 3 개. </li></ol> 5. 배양 마우스 구개 선반 <ol><li> tissu을 품어72 시간 동안 가습 가스 혼합물 (5 % CO 2, 95 % 공기)에서 37 ° C에서 ES, 그리고 매체마다 24 시간을 변경합니다. </li></ol> 조직 학적 분석 6. 미각 처리 <ol><li> / N은 ​​4 °에서 4 % 포름 알데히드 / 인산염 완충 생리 식염수 O와 문화에 72 시간 후 입맛을 수정 &#160; C. 그런 다음, 부드럽게 전송 피펫 PBS로 배양 접시에 조직을 씻는다. 카세트를 포함에 실험실 와이프과 장소의 조각에 입맛을 감싸. 참고 : 실험실 와이프의 입맛 포장은 탈수 과정에서 자신의 손실을 방지 할 수 있습니다. </li><li> 70 %, 80 % 및 다음과 같은 정규 파라핀 매립 절차이어서 1 시간 동안 100 % 에탄올 세척, 최대 조직 탈수. 내장의 경우, 트레이에 직면 미각의 앞쪽 부분을 놓습니다. </li><li> 전치부에서 관상 방향으로 직렬 6 μm의 섹션을 잘라. 전체 섹션 미각은 350-450 섹션에 ì을 얻을 것입니다기능. 헤 마톡 실린 및 에오신 (H &amp; E). 10 점수 앞서 설명한 평균 퓨전 점수를 사용하여 매 20 번째 섹션 (MFS) 규모 (표 1)와 섹션을 얼룩. (8) </li></ol>

<ol>
	<li>Nawshad, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palatal+seam+disintegration+to+die+or+not+to+die+that+is+no+longer+the+question.">Palatal seam disintegration to die or not to die that is no longer the question.</a> Developmental dynamics an official publication of the American Association of Anatomists. 237, 2643-2656 (2008).</li><li>Strong, E. B., Buckmiller, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+the+cleft+palate.">Management of the cleft palate.</a> Facial plastic surgery clinics of North America. 9, 15-25 (2001).</li><li>Witt, P. D., Marsh, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+assessing+outcome+of+surgical+repair+of+cleft+lip+and+cleft+palate.">Advances in assessing outcome of surgical repair of cleft lip and cleft palate.</a> Plastic and reconstructive surgery. 100, 1907-1917 (1997).</li><li>4miloro, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> principles of oral and maxillofafcial surgery. 209, 231-249 (1984).</li><li>Mima, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+epithelial+adhesion+molecule+CEACAM1+is+important+for+palate+formation.">Regulation of the epithelial adhesion molecule CEACAM1 is important for palate formation.</a> PloS one. 8, e61653 (2013).</li><li>Carette, M. J., Ferguson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fate+of+medial+edge+epithelial+cells+during+palatal+fusion+in+vitro+an+analysis+by+DiI+labelling+and+confocal+microscopy.">The fate of medial edge epithelial cells during palatal fusion in vitro an analysis by DiI labelling and confocal microscopy.</a> Development (Cambridge, England). 114, 379-388 (1992).</li><li>Ferguson, M. W., Honig, L. S., Slavkin, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+cultured+palatal+shelves+from+alligator+chick+and+mouse+embryos.">Differentiation of cultured palatal shelves from alligator chick and mouse embryos.</a> The Anatomical record. 209, 231-249 (1984).</li><li>San Miguel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ephrin+reverse+signaling+controls+palate+fusion+via+a+PI3+kinase+dependent+mechanism.">Ephrin reverse signaling controls palate fusion via a PI3 kinase dependent mechanism.</a> Developmental dynamics an official publication of the American Association of Anatomists. 240, 357-364 (2011).</li><li>Kang, P., Svoboda, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI+3+kinase+activity+is+required+for+epithelial+mesenchymal+transformation+during+palate+fusion.">PI 3 kinase activity is required for epithelial mesenchymal transformation during palate fusion.</a> Developmental dynamics an official publication of the American Association of Anatomists. 225, 316-322 (2002).</li><li>RD, 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> Histopathologic Technic and Practical Histochemistry. , (1965).</li><li>Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Atlas of Mouse Development. , (1992).</li><li>Taher, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+gene+expression+analysis+of+murine+limb+development.">Global gene expression analysis of murine limb development.</a> PloS one. 6, e28358 (2011).</li></ol>

The authors do not have any competing financial interests.

이 문서의 프로토콜은 배아 일 13.5에서 배아에서 구개 선반을 해부하는 방법을 제공한다. 마우스 배아 구개 가구는 37 ° C에서 95 % O 2, 5 % CO 2 분위기하에 무 혈청 배지에서 배양 하였다. 성공적인 구개 해부는 문화의 완료에 안락사 쥐의 배아 시간의 절차를 수행하는 동안 각 단계에서 여러 요인에 결정적으로 의존한다. 구개 융합을 좌우하는 가장 중요한 요소 중 하나는 기관 배양...

구강 안면 쪼개진 조각은 가장 지배적 인 두개 안면 기형이다. 또한, 고려 모든 가능한 두개 안면 결함을 가지고,이 신생아 2에서 두 번째로 가장 흔한 선천성 기형이다. 구개 파열은 약 1 미국 (US) 매년마다 700 출생에서 발생, 구개열의 발생 빈도는 갈라진 달에 입맛 또는 3 일 당 쪼개진 조각 15 아이들과 함께 탄생 475 어린이와 동일하다. 어린이의 약 1 %가 매년 전시를 전세계 두개 안면 dysmorphology 어떤 형태의 탄생. 입천장과 입술의 갈라진 틈이 이상이 환자의 평생의 의미와 매우 비싸고 복잡한 절차가 필요합니다. 구강 갈라진 각 환자에 대한 예상 비용은 약 $ 100,000 4입니다. 구순 구개열 환자의 치료는, 두개 안면 외과, 이비인후과, 유전 학자, 마취를 포함하여 의사의 팀이 필요합니다음성 언어 병리학 자, 영양사, 치열 교정, 보철, 심리학자, 신경 외과 및 안과. palatogenesis에서 보조 구개는 처음에 수직으로 성장하고 혀의 배부 위에 구개 선반 높이를 받아야 쌍의 outgrowths로 발생한다. 해발 고도에 따라, 한 쌍의 구개 선반 (인간의 마우스에서 E14.5 -E15에서와 주 9) 중간 선을 향해 성장한다. 선반 팁을 포함 내측 가장자리 상피 (MEE)는 중간 선 상피 솔기를 형성 준수합니다. 이것은 간엽 합류 할 수 있도록 중간 엽 전이 및 / 또는 아폽토시스에 의해 상피 이어진다. 반대 MEE의 접착은 그 변경 구개 파열을 일으키는 중요한 이벤트입니다. 그러나, 몇몇 연구는 구개 ​​선반 (5)의 초기 접착력을 조사 하였다. 조골 세포로 중간 엽 세포의 분화에 의해 하드 구개 형성한다. 음자리표를 생산할 수있는 미각의 이상 개발T와 함께 또는 입술의 개입없이 입맛. 미각 기관 배양 기술은 지난 30 년 동안 많은 실험실 -6,7- 위해 널리 사용되었다. 이 프로토콜에서 우리는 세부 사항에 구개 해부 및 정적 기관 배양하는 방법을 설명합니다. 움직 기관 배양의 장점은 선반 구개 융합 할 수 있다는 것이다. 이 기술은 많은 융합 및 신호 실험 8,9에 대한 우리의 실험실에서 성공적으로 사용되었다. 기술의 범위는 광대 한 정적 기관 배양 물 시스템은 외인성 화학 물질에 대한 반응의 평가를 포함하여, 필요할 때마다 이용 될 수 있지만, 다른 경로 및 특정 단백질 규제 성장 인자의 효과. <img alt="그림 1" src="/files/ftp_upload/53063/53063fig1.jpg" /> 그림 1. 마우스 Palatogenesis. 쥐의 미각의 발달 단계. (BF) 스캔 ELE대표 발달 시간에 차 구개의 ctron 현미경 (SEM). 빨간색 화살표 : 구개 부착 및 융합의 초기 부분을 보여줍니다. 노란색 화살표 : 융합 후 사라집니다 기본 및 보조 입맛 사이의 공간에 포인트 (PLoS의 하나의 허가 카우프만 (11)에서 재 인쇄).

<table><tbody><tr><td>BGJ&lt;sub&gt;b&lt;/sub&gt;</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Lonza Inc.</td><td>17-602E</td><td>100X</td></tr><tr><td>Petri dishes</td><td>VWR</td><td>25384-088</td><td>100 x15 mm</td></tr><tr><td>Center-well organ culture dish</td><td>Falcon&amp;nbsp;</td><td>#353037</td><td>60 x15 mm</td></tr><tr><td>Wire triangular grid</td><td>Custom made with stainless steel wire mesh to fit the organ culture dish well.&amp;nbsp;&amp;nbsp;</td><td></td><td></td></tr><tr><td>Polycarbonate filter</td><td>GE&amp;amp; Water and Process Technologies</td><td>K04BP04700</td><td>Black 0.4 micron, 47 mm</td></tr><tr><td>Stereo microscope</td><td>ZEISS&amp;nbsp; Stemi SR</td><td></td><td></td></tr><tr><td>Culture hood</td><td>NUAIRE Laminar Flow</td><td></td><td></td></tr><tr><td>Microdissecting forceps</td><td>Dumont Medical&amp;nbsp;</td><td></td><td>#5</td></tr><tr><td>Microdissecting scissors</td><td>Kent Scientific Corporation</td><td>INS14003-G</td><td>Vannas Scissors, straight, 8 cm long, 0.1mm tips, 5 mm blades, German made</td></tr><tr><td>Microdissecting scissors</td><td>Kent Scientific Corporation</td><td>INS500086</td><td>Vannas Scissors, straight, 8.5 cm long, 0.025 mm x 0.015 mm tips, 7 mm blades</td></tr><tr><td>Microdissecting scissors</td><td>Kent Scientific Corporation</td><td>INS14127-G</td><td>Spring scissors curved curved, 10.5 cm long, 8 mm blades, German made</td></tr><tr><td>Surgical currete (spoon spatula)</td><td>Hu-friedy</td><td></td><td>CM 2/4</td></tr><tr><td>Protector laboratory hood</td><td>Labconco</td><td></td><td></td></tr><tr><td>Incubator</td><td>Thermo Scientific</td><td></td><td>Farma</td></tr><tr><td>Series11 water Jacket</td><td></td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td></td><td></td><td></td></tr><tr><td>PBS&amp;nbsp;</td><td>GIBCO</td><td>10010-023&amp;nbsp;</td><td>1X</td></tr><tr><td>Fiber optic Light source</td><td>Fiber-light&amp;nbsp; Dolan-Jenner</td><td>PL-750</td><td></td></tr><tr><td>Embedding cassette&amp;nbsp;</td><td>Statlab</td><td>EC301</td><td></td></tr><tr><td>Kim Wipes</td><td>VWR</td><td>470173-504</td><td></td></tr></tbody></table>

method of studying palatal fusion using static organ culture

구순 구개열은 모든 출생 결함의 가장 흔한 중입니다. 중간 선 상피 솔기를 형성하기 위해 부착 상피로 덮여 중간 엽 선반에서 보조 구개 형태​​ (MES). 이론은 MES 세포 융합 미각 (1)을, 중간 엽 전이 (EMT), 세포 사멸 및 마이그레이션 상피를 수행하는 것이 좋습니다. MES의 전체 붕괴는 중간 엽 세포를 둘러싼 구개 합류의 마지막 필수 단계입니다. 우리는 구개 장기 배양 방법을 제공한다. 체외 프로토콜 개발이 융합 동안 생물학적 및 분자 프로세스의 연구를 할 수 있습니다. 이 기술의 응용 프로그램은 외인성 화학 약품에 대한 평가 응답, 규제 및 성장 요인과 특정 단백질의 효과를 포함, 수많은 있습니다. 구개 기관 배양 물이 생체 내 연구에서 사용 불가능 개발의 다른 단계에서의 조작을 포함하여 다수의 이점을 갖는다.

기술이 연구 experiments.The 다음의 다른 종류에 적용될 수는 구개 융합에 체외 니코틴 기형 효과를 시험하기 위해 설계되었다. 마우스의 두 균주는이 실험에 이용 된, CD1 및 C57.Palatal 조직 니코틴 헤미 0.06 및 6 mM의 농도로 처리 하였다. 그것은 구개 융합 패턴 및 타이밍이 서로 다른 두 균주에서 다르다는 것을 관찰 하였다. CD1 쥐에서, 대조군 구개 가구는 시간 의존적 방식으로 융합을 계속했?...

Watch this Scientific Journal Video about Method of Studying Palatal Fusion using Static Organ Culture at JoVE.com

Method of Studying Palatal Fusion using Static Organ Culture

구개 개발의 연구는 구개열, 엄청난 의료 부담을 부과하고 외관을 손상하는 지속 남길 수 있습니다 출생 결함의 발생에 의해 좌우된다. 우리는 여기서 구개 개발 및 융합에 관련된 다른 신호 경로를 연구하기 위해 사용될 수있다 배양 구개 선반 기술을 보여준다.

정적 장기 문화를 사용하여 구개 퓨전 공부의 방법

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that ...

method-of-studying-palatal-fusion-using-static-organ-culture

Research

JoVE Journal

Developmental Biology

8.7K 조회수.  Texas A&M University Baylor College of Dentistry.  구개 개발의 연구는 구개열, 엄청난 의료 부담을 부과하고 외관을 손상하는 지속 남길 수 있습니다 출생 결함의 발생에 의해 좌우된다. 우리는 여기서 구개 개발 및 융합에 관련된 다른 신호 경로를 연구하기 위해 사용될 수있다 배양 구개 선반 기술을 보여준다. 

비디오: Method of Studying Palatal Fusion using Static Organ Culture

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

인두 아치 전의 VIVO 문화는 심장과 근육 전구 세포와 그 틈새를 연구

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

연구

발생학

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

형광에 원위치 하이브리드화 및 공촛점 레이저 스캐닝 현미경에 의해 맛이 좋은 확장기의 사정 Biofilm 분석

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

의학

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

인간 Fungiform의 맛 용의자 전지의 분리 및 문화

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

신경과학

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Mouse Secondary Palate Fusion의 라이브 이미징

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

We present a protocol for isolation and culture of primary mouse embryonic palatal mesenchymal cells for time-lapse imaging of two-dimensional (2D) growth and wound-repair assays. We also provide the methodology for analysis of the time-lapse imaging data to determine cell-stream formation and directional motility.

집단 운동 특성을 분석하기 위해 1차 마우스 배아 성 성 메센키메 세포의 격리 및 시간 경과 이미징

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and ...

Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is powered by local progenitor cells and renders taste function prone to disruption by a multitude of medical treatments, which in turn severely impacts the quality of life. Thus, studying this process in the context of drug treatment is vital to understanding if and how taste progenitor function and TRC production are affected. Given the ethical concerns and limited availability of human taste tissue, mouse models, which have a taste system similar to humans, are commonly used. Compared to in vivo methods, which are time-consuming, expensive, and not amenable to high throughput studies, murine lingual organoids can enable experiments to be run rapidly with many replicates and fewer mice. Here, previously published protocols have been adapted and a standardized method for generating taste organoids from taste progenitor cells isolated from the circumvallate papilla (CVP) of adult mice is presented. Taste progenitor cells in the CVP express LGR5 and can be isolated via EGFP fluorescence-activated cell sorting (FACS) from mice carrying an Lgr5EGFP-IRES-CreERT2 allele. Sorted cells are plated onto a matrix gel-based 3D culture system and cultured for 12 days. Organoids expand for the first 6 days of the culture period via proliferation and then enter a differentiation phase, during which they generate all three taste cell types along with non-taste epithelial cells. Organoids can be harvested upon maturation at day 12 or at any time during the growth process for RNA expression and immunohistochemical analysis. Standardizing culture methods for production of lingual organoids from adult stem cells will improve reproducibility and advance lingual organoids as a powerful drug screening tool in the fight to help patients experiencing taste dysfunction.

The protocol presents a method for culturing and processing lingual organoids derived from taste stem cells isolated from the posterior taste papilla of adult mice.

성인 마우스 맛 줄기 세포에서 파생 된 언어 오르가노이드의 생성 및 배양

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is ...

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The continuously growing mouse incisor provides an excellent model to study tissue-specific stem cells. The epithelial tissue-specific stem cells of the incisor are located at the proximal end of the tooth in a niche called the cervical loop. They provide a continuous influx of cells to counterbalance the constant abrasion of the self-sharpening tip of the tooth. Presented here is a detailed protocol for the isolation and culture of the proximal end of the mouse incisor that houses stem cells and their niche. This is a modified Trowell-type organ culture protocol that enables in vitro culture of tissue pieces (explants), as well as the thick tissue slices at the liquid/air interface on a filter supported by a metal grid. The organ culture protocol described here enables tissue manipulations not feasible in vivo, and when combined with the use of a fluorescent reporter(s), it provides a platform for the identification and tracking of discrete cell populations in live tissues over time, including stem cells. Various regulatory molecules and pharmacological compounds can be tested in this system for their effect on stem cells and their niches. This ultimately provides a valuable tool to study stem cell regulation and maintenance.

The Trowell-type organ culture method has been used to unravel complex signaling networks that govern tooth development and, more recently, for studying regulation involved in stem cells of the continuously growing mouse incisor. Fluorescent-reporter animal models and live-imaging methods facilitate in-depth analyses of dental stem cells and their specific niche microenvironment.

치아 줄기 세포의 조절 연구를 위한 트로웰형 장기 배양 사용

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The ...

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

곰팡이, 둘레 및 미각 미각의 전체 마운트 염색, 시각화 및 분석

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

인간 비강 상피 오가노이드의 문화와 이미징

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

성인 마우스 입천장에서 1 차 구강 각막 세포의 격리 및 문화

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...