Z.C. 인정 다트머스 시작 기금과 브 랑 쿠와 이즈-과학 원정대, ETH 쮜 리 히에 의해 관리에 대 한 사회에서 지원 합니다. 저자는 박사 래리 A. Taber, Benjamen A. Filas, Qiaohang Guo Yunfei 시 유용한 토론에 대 한 뿐만 아니라 익명 검토자 의견 감사합니다. 이 자료는 국립 과학 재단 대학원 연구 친교 그랜트 번호 아래에서 지 원하는 작업 기반 DGE-1313911입니다. 모든 의견, 결과, 결론 또는 권고가이 자료에서 표현은 저자 (들)의 고 반드시 국립 과학 재단의 의견을 반영 하지 않습니다.

1입니다. 조직 배양의 준비
<ol><li>문화 미디어에 대 한 기반으로 4.5 g/L 포도 당, 나트륨 중 탄산염, 그리고 L-글루타민 0.5 L 병 Dulbecco의 수정이 글의 중간 (DMEM)의 사용 합니다.</li>
	<li>살 균 층 흐름 후드에서 DMEM의 0.5 l 항생제의 10 mL를 추가 합니다.</li>
	<li>살 균 피 펫을 사용 하 여 살 균 50 mL 원뿔 튜브 DMEM 항생제 솔루션 50 mL 전송.</li>
	<li>살 균 후드에 0.5 L 병에서 나머지 DMEM 항생제 솔루션에 병아리 세럼 50 mL를 추가 합니다.</li>
	<li>(라고도 약칭 병아리 문화 미디어 [CCM])-20 ° c.에 50 mL 원뿔 튜브 aliquots에 최종 솔루션 저장</li>
</ol>2. 달걀 부 화
<ol><li>섬세 한 사용을 70% 에탄올 수정된 특정 병원 체 자유로운 백색 레그 혼 닭 계란을 청소 정리 합니다. 계란 홀더에 세로 방향으로 정렬 합니다.</li>
	<li>37.5 ° C의 목표 온도 설정 하 고 48-55%에 습도 유지 하는 계란 부 화기를 켭니다. 습도 인큐베이터에 물 적절 한 양의 추가 하 여 제어 됩니다.</li>
	<li>HH11-13, 40-44 h 약을 알을 품 어.</li>
	<li>계란 살포 / 70% 에탄올으로 청소 하기 전에 약 15-30 분 동안 실내 온도에 냉각 하자.</li>
</ol>3. 풀 유리 모 세관
<ol><li>Micropipette 끌어당기는 사람에는 외부 직경 1.0 m m와 0.5 m m 내부 직경 10 cm 길이의 유리 모 세관 튜브를 탑재 합니다.</li>
	<li>열을 설정 하 고 각각 750와 400, 매개 변수를 당겨. 얇은 바늘으로 모 세관 튜브를 당겨 단추를 누릅니다.</li>
</ol>4. 필터 종이 캐리어 메서드
<ol><li>필터 종이에서 직경에서 대략 3 cm의 동그라미를 잘라.</li>
	<li>사각형 약 1cm를 잘라 2cm 구멍 구멍을 뚫는 사용 하 여 원형에서. 어떤 돌기 제거 또는 날카로운 모서리를 있는지 확인 하십시오.</li>
</ol>5. 배아 수확 및 준비
<ol><li>바닥에서 계란 균열, 당겨 떨어져 껍질 부드럽게 하 고 신중 하 게 150 m m x 15 mm 페 트리 접시에 내용을 입금. 태아 측이, 그들은 그들을 크래킹 하는 동안 incubated 했다 동일한 방향에서 계란을 계속 합니다.</li>
	<li>일회용 파스퇴르 피 펫을 사용 하 여 얇은 알 부 민을 제거 합니다.</li>
	<li>무뚝뚝한 끝난된 집게를 사용 하 여 노 른 자에서 두꺼운 알 부 민을 구분 합니다. 가볍게 무뚝뚝한 끝난된 집게와 노른자위의 상단 된다고 하 여 두꺼운 알 부 민 제거 되었습니다 확인 하십시오.</li>
	<li>뾰족한 집게를 사용 하 여 센터와 배아, 태아의 긴 축과 링의 긴 축에 일치 하는 필터 종이 반지 장소.</li>
	<li>가 위로 필터 종이 반지를 둘러싼 노 른 자를.</li>
	<li>노 른 자 먼저 렸 어디 사이트 쪽으로 비스듬한 방향에서 반지 및 노른자위에서 배아를 당겨.</li>
	<li>실내 온도 1 버퍼링 하는 인산 염 (PBS) x 두 순차 100 mm 요리에 배아를 린스.</li>
	<li>35 mm 페 트리 접시에 먼저 필터 종이 반지를 놓습니다. 다음 장소 배아 등 쪽을 필터 종이에 이미 35 mm 페 트리 접시에.</li>
	<li>장소 필터 종이 샌드위치 위에 스테인리스 반지. 태아 손상 되었는지 확인 합니다.</li>
	<li>각 페 트리 접시를 이전 준비 ccm 3 mL를 추가 합니다.</li>
	<li>가볍게 태아와 멀리 VM을 필 링, (바로 위에 개) 앞쪽 끝에서 시작 및 notochord (그림 1A)를 진행 상단 가져온된 모 세관 바늘을 감추고 여 각 배아의 VM을 제거 합니다.</li>
	<li>(습도 유지)을 물 포화 섬세 한 작업 잎사귀 줄지어 했다 한 150 mm 접시에 8 35 mm 페 트리 접시를 놓습니다.</li>
	<li>장소 sealable 비닐 봉지에 150 mm 페 트리 접시 다음 95% O2 , 5% CO2를 이루어져 있는 가스 혼합물으로 가방을 채우십시오.</li>
	<li>가방을 봉인 하 고 37.5 ° C 인큐베이터에 배치.</li>
	<li>HH15-HH16 (그림 1B)까지 추가 27 h에 대 한 태아를 품 어.</li>
</ol>6. 유도 표면 장력
<ol><li>인큐베이터에서 배아를 제거 하 고 그들의 이미지를 광학 일관성 단층 촬영 (OCT) 시스템을 사용 하 여. 신경 관 (NT) (그림 2)의 비틀림 각도를 사용 하 여 10 월</li>
	<li>가벼운 현미경에 배아를 전송 하 고 10 배 확대에서 시각화. 200 microliter 피 펫을 사용 하 여 점진적으로 배양 접시에서 미디어의 0.2 mL를 제거.</li>
	<li>태아에는 미디어에 어 인터페이스의 효과 관찰 하는 각 간격에서 명시 이미지를 가져가 라.</li>
	<li>태아에 걸쳐 표면 장력 비틀림 (그림 1C) 유도 될 때까지 미디어를 제거 합니다.</li>
	<li>배아 배아를 제어 하는 비교에 대 한 최종 비틀림 각도 설정 하려면 다시 한번 OCT 시스템을 사용 하 여 이미지.  참고: 밝은 필드 이미지 해 현미경을 사용 하 여 인수 했다. 광학 일관성 단층 촬영 시스템 연결 된 현미경으로 살아있는 배아의 단면 이미지 스택을 얻으려고 사용 되었다. 이미지 3 x 10 m m x 3 m m3 검색 도메인에서 얻은 다음 이미징 소프트웨어에서 처리. 마지막으로, 실제 모델 이미지는 디지털 싱글 렌즈 리 플렉스 카메라와 함께 찍은 사진.</li>
</ol>7. 표면 장력/VM 세력의 물리적 모델링
<ol><li>상업적인 모델링 소프트웨어 (그림 3A) HH14-17 사이의 배아 유사한 단순화 된 3D 형상을 개발 합니다.</li>
	<li>상업적인 3D 컴퓨터 그래픽 소프트웨어에서 3D 형상의 부정적인 형 디자인.</li>
	<li>3D 프린터 로드 1.75 m m 아크릴로 니트 릴 부 타 디 엔 스틸 렌 필 라 멘 트 3D 사용 하 여 인쇄 stereolithographic (.stl) 형태로 설계 된 금형.</li>
	<li>캐스팅 금형, 실리콘 고무 탄성 중합체 부품 A와 같은 부분에 B를 혼합 하 고 즉시; 금형에 혼합물을 부 어 12 h (그림 3B)에 대 한 실 온에서 치료 주조 금형을 설정 합니다.</li>
	<li>비틀림을 시각화 하는 등 측면에서 NT 따라 배아의 실제 모델을 표시 합니다.</li>
	<li>VM 또는 표면 장력 (그림 3D)의 모방을 3 차원 물리 모델에 적용 하는 힘을 복제 하는 coverslip를 사용 합니다.</li>
	<li>물리적 모델의 측면 길이가 엄밀한 전선의 시리즈를 삽입 합니다. 후는 coverslip 뇌 모델에 외부의 힘을 미치는, 전선 될 위치에 따라 각도에서 기운 다. 각 와이어 (그림 3E)의 원래 길이에 의해 arctan 예상된 길이의 회전 각도 결정 합니다.</li>
</ol>8. 심장 루프의 방향 변경
<ol><li>3.1 및 3.2 가져온된 모 세관 튜브를 단계를 따릅니다.</li>
	<li>5.1 5.10 태아 준비를 통해 단계를 따릅니다.</li>
	<li>집게의 쌍을 사용 하 여 태아 복 부-사이드 업 되도록 필터 종이 플립.</li>
	<li>가져온된 모 세관 튜브를 사용 하 여 splanchnopleure (SPL) 막에 슬릿 오픈 컷.</li>
	<li>모 세관 튜브를 사용 하 여 왼쪽을 오른쪽에서 마음을 밀어 기계적인 힘을 발휘 하.</li>
	<li>5.12 5.15 비틀림의 변화를 관찰 하는 것을 통해의 단계를 따릅니다.</li>
</ol>

<ol>
	<li>Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+of+Growth,+Remodeling,+and+Morphogenesis.">Biomechanics of Growth, Remodeling, and Morphogenesis.</a> Appl. Mech. Rev. 48, 487-545 (1995).</li><li>Wyczalkowski, M. A., Chen, Z., Filas, B. A., Varner, V. D., Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+models+for+mechanics+of+morphogenesis.">Computational models for mechanics of morphogenesis.</a> Birth Defects Research Part C - Embryo Today: Reviews. 96, 132-152 (2012).</li><li>Savin, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+growth+and+form+of+the+gut.">On the growth and form of the gut.</a> Nature. 476, 57-62 (2011).</li><li>Gjorevski, N., Nelson, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanics+of+development:+Models+and+methods+for+tissue+morphogenesis.">The mechanics of development: Models and methods for tissue morphogenesis.</a> Birth Defects Research Part C - Embryo Today: Reviews. 90, 193-202 (2010).</li><li>Shyer, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Villification:+how+the+gut+gets+its+villi.">Villification: how the gut gets its villi.</a> Science. 342, 212-218 (2013).</li><li>Ambrosi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+biological+growth+and+remodeling.">Perspectives on biological growth and remodeling.</a> Journal of the Mechanics and Physics of Solids. 59, 863-883 (2011).</li><li>Chen, Z., Majidi, C., Srolovitz, D. J., Haataja, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+helical+ribbons.">Tunable helical ribbons.</a> Appl. Phys. Lett. 98, (2011).</li><li>Gerbode, S. J., Puzey, J. R., McCormick, A. G., Mahadevan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+cucumber+tendril+coils+and+overwinds.">How the cucumber tendril coils and overwinds.</a> Science. 337, 1087-1091 (2012).</li><li>Armon, S., Efrati, E., Kupferman, R., Sharon, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometry+and+mechanics+in+the+opening+of+chiral+seed+pods.">Geometry and mechanics in the opening of chiral seed pods.</a> Science. 333, 1726-1730 (2011).</li><li>Filas, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+potential+role+for+differential+contractility+in+early+brain+development+and+evolution.">A potential role for differential contractility in early brain development and evolution.</a> Biomech. Model. Mechanobiol. 11, 1251-1262 (2012).</li><li>Xu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axons+pull+on+the+brain,+but+tension+does+not+drive+cortical+folding.">Axons pull on the brain, but tension does not drive cortical folding.</a> J. Biomech. Eng. 132, 71013 (2010).</li><li>Chen, Z., Guo, Q., Dai, E., Forsch, N., Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+embryonic+chick+brain+twists.">How the embryonic chick brain twists.</a> J. R. Soc. Interface. 13, (2016).</li><li>Manca, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+growth+factor+regulates+axial+rotation+during+early+stages+of+chick+embryo+development.">Nerve growth factor regulates axial rotation during early stages of chick embryo development.</a> Proc Natl Acad Sci U S A. 109, 2009-2014 (2012).</li><li>Shi, Y., Yao, J., Xu, G., Taber, L. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bending+of+the+looping+heart:+differential+growth+revisited.">Bending of the looping heart: differential growth revisited.</a> J. Biomech. Eng. 136, 1-15 (2014).</li><li>Shi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bending+and+twisting+the+embryonic+heart:+A+computational+model+for+c-looping+based+on+realistic+geometry.">Bending and twisting the embryonic heart: A computational model for c-looping based on realistic geometry.</a> Front. Physiol. 5, (2014).</li><li>Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphomechanics:+Transforming+tubes+into+organs.">Morphomechanics: Transforming tubes into organs.</a> Current Opinion in Genetics and Development. 27, 7-13 (2014).</li><li>Hosseini, H. S., Beebe, D. C., Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+effects+of+the+surface+ectoderm+on+optic+vesicle+morphogenesis+in+the+chick+embryo.">Mechanical effects of the surface ectoderm on optic vesicle morphogenesis in the chick embryo.</a> J. Biomech. 47, 3837-3846 (2014).</li><li>Hamburger, V., Hamilton, H. 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+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Dev. Dyn. 88, 49-92 (1951).</li><li>Shi, Y., Varner, V. D., Taber, L. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+is+cytoskeletal+contraction+required+for+cardiac+fusion+before+but+not+after+looping+begins?.">Why is cytoskeletal contraction required for cardiac fusion before but not after looping begins?.</a> Phys. Biol. 12, 16012 (2015).</li><li>Garcia, K. E., Okamoto, R. J., Bayly, P. V., Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contraction+and+stress-dependent+growth+shape+the+forebrain+of+the+early+chicken+embryo.">Contraction and stress-dependent growth shape the forebrain of the early chicken embryo.</a> J. Mech. Behav. Biomed. Mater. 65, 383-397 (2017).</li><li>Faisst, A. M., Alvarez-Bolado, G., Treichel, D., Gruss, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rotatin+is+a+novel+gene+required+for+axial+rotation+and+left-right+specification+in+mouse+embryos.">Rotatin is a novel gene required for axial rotation and left-right specification in mouse embryos.</a> Mech. Dev. 113, 15-28 (2002).</li><li>Yalcin, H. C., Shekhar, A., Rane, A. a., Butcher, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex-ovo+chicken+embryo+culture+system+suitable+for+imaging+and+microsurgery+applications.">An ex-ovo chicken embryo culture system suitable for imaging and microsurgery applications.</a> J. Vis. Exp. (44), (2010).</li><li>Chapman, S. C., Collignon, J., Schoenwolf, G. C., Lumsden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+method+for+chick+whole-embryo+culture+using+a+filter+paper+carrier.">Improved method for chick whole-embryo culture using a filter paper carrier.</a> Dev. Dyn. 220, 284-289 (2001).</li><li>Camp&#224;s, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+cell-generated+mechanical+forces+within+living+embryonic+tissues.">Quantifying cell-generated mechanical forces within living embryonic tissues.</a> Nat. Methods. 11, 183-189 (2014).</li><li>Schierenberg, E., Junkersdorf, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+eggshell+and+underlying+vitelline+membrane+for+normal+pattern+formation+in+the+early+C.+elegans+embryo.">The role of eggshell and underlying vitelline membrane for normal pattern formation in the early C. elegans embryo.</a> Roux's Arch. Dev. Biol. 202, 10-16 (1992).</li><li>Chuai, M., Weijer, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mechanisms+Underlying+Primitive+Streak+Formation+in+the+Chick+Embryo.">The Mechanisms Underlying Primitive Streak Formation in the Chick Embryo.</a> Current Topics in Developmental Biology. 81, 135-156 (2008).</li><li>Voronov, D. A., Alford, P. W., Xu, G., Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mechanical+forces+in+dextral+rotation+during+cardiac+looping+in+the+chick+embryo.">The role of mechanical forces in dextral rotation during cardiac looping in the chick embryo.</a> Dev. Biol. 272, 339-350 (2004).</li><li>Raya, A., Izpisua Belmonte, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unveiling+the+establishment+of+left-right+asymmetry+in+the+chick+embryo.">Unveiling the establishment of left-right asymmetry in the chick embryo.</a> Mechanisms of Development. 121, 1043-1054 (2004).</li><li>Voronov, D. A., Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+looping+in+experimental+conditions:+Effects+of+extraembryonic+forces.">Cardiac looping in experimental conditions: Effects of extraembryonic forces.</a> Dev. Dyn. 224, 413-421 (2002).</li><li>Chen, Z., Huang, G., Trase, I., Han, X., Mei, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+Self-Assembly+of+a+Strain-Engineered+Flexible+Layer:+Wrinkling,+Rolling,+and+Twisting.">Mechanical Self-Assembly of a Strain-Engineered Flexible Layer: Wrinkling, Rolling, and Twisting.</a> Phys. Rev. Appl. 5, (2016).</li><li>Manner, J., Seidl, W., Steding, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+cervical+flexure:+an+experimental+study+on+chick+embryos.">Formation of the cervical flexure: an experimental study on chick embryos.</a> Acta Anat. (Basel). 152, 1-10 (1995).</li><li>Ware, M., Schubert, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+early+axon+scaffold+in+the+rostral+brain+of+the+chick+embryo.">Development of the early axon scaffold in the rostral brain of the chick embryo.</a> J. Anat. 219, 203-216 (2011).</li><li>Ramasubramanian, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+role+of+intrinsic+and+extrinsic+forces+in+early+cardiac+S-looping.">On the role of intrinsic and extrinsic forces in early cardiac S-looping.</a> Dev. Dyn. 242, 801-816 (2013).</li><li>Hoyle, C., Brown, N. a., Wolpert, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+left/right+handedness+in+the+chick+heart.">Development of left/right handedness in the chick heart.</a> Development. 115, 1071-1078 (1992).</li><li>Nerurkar, N. L., Ramasubramanian, A., Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenetic+adaptation+of+the+looping+embryonic+heart+to+altered+mechanical+loads.">Morphogenetic adaptation of the looping embryonic heart to altered mechanical loads.</a> Dev. Dyn. 235, 1822-1829 (2006).</li><li>Levin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left-right+asymmetry+and+the+chick+embryo.">Left-right asymmetry and the chick embryo.</a> Semin. Cell Dev. Biol. 9, 67-76 (1998).</li><li>Roebroek, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Failure+of+ventral+closure+and+axial+rotation+in+embryos+lacking+the+proprotein+convertase+Furin.">Failure of ventral closure and axial rotation in embryos lacking the proprotein convertase Furin.</a> Development. 125, 4863-4876 (1998).</li><li>Peebles, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+proton+spectroscopy+and+diffusion+weighted+imaging+of+chick+embryo+brain+in+ovo.">Magnetic resonance proton spectroscopy and diffusion weighted imaging of chick embryo brain in ovo.</a> Dev. Brain Res. 141, 101-107 (2003).</li><li>Zhu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerberus+regulates+left-right+asymmetry+of+the+embryonic+head+and+heart.">Cerberus regulates left-right asymmetry of the embryonic head and heart.</a> Curr. Biol. 9, 931-938 (1999).</li></ol>

저자는 관심 없음 충돌 선언합니다.

반면 물리적 현상 morphogenesis26,27,,2829,30, 특정 기계 메커니즘과 함께 기계 조정에에서 중요 한 역할 및 분자 메커니즘, 크게 미개척 남아 있습니다. 그것은 복 부 굴곡와 원시적인 뇌의 우 비틀림 초기 배아 morphogenesis18,31,<su...

현대 개발 생물학 연구는 주로 분자 유전학1,2,,34,,56 의 관점에서 이해 개발에 초점을 맞추고합니다 , 7 , 8 , 9 , 10 , 11 , 12 , 13. 현상과 morphogenesis에서 중심 역할을 담당할 또는 생물의 생성 형성14,15,,1617; 알려져 있다 그러나, 개발의 특정 기계적 메커니즘 크게 unstudied 남아 있다. 복 부 굴곡 및 원시 뇌 관 후 햄버거 해밀턴 무대 11 (HH 11)18 배아 모양에 기여 하는 두 가지 주요 프로세스의 우 비틀림19,20을 변경 합니다. 특히, 배아 두뇌에 비틀림 개발 기본 물리적 메커니즘 불완전 하 게 이해 남아 있습니다.
병아리 태아에 배아 비틀림 개발에 왼쪽 오른쪽 (L R) 비대칭의 초기 전체적 이벤트 중 하나입니다. L-R 비대칭의 과정을 교란 하는 경우 사이트 inversus, 이성질체또는 heterotaxia 같은 출생 결함21을발생 합니다. 여기 선물이 전 비켜 실험22,23 물리적 모델링 초기 미 발달 두뇌 개발 하는 동안 기계적인 힘의 특성을 결합 하는 프로토콜. 제시 하는 방법의 목표는 기계적인 힘 두뇌와 초기 개발12동안 비틀림의 정도 영향을 주는 요인에 대 한 책임을 식별 하는입니다. Vitelline 막 (VM) 태아의 등 쪽을 구속 하는 실험 관찰을 바탕으로, 우리는 VM 개발 두뇌의 비틀림을 유도 하는 데 필요한 힘을 제공 한다는 가설. 따라서,이 방법에서는, 두뇌 염력에 효과를 뇌 영역을 커버 하는 VM의 일부를 제거 했습니다. 또한, 액체의 표면 장력을 적용 하는 방법은 VM의 기계적 역할을 확인 하 고 이전 수행 하지 않았다면 뇌 염력에 필요한 힘의 견적을 제공 하 사용 되었다. 배아 morphogenesis 동안 힘을 측정 하는 것은 어려운 작업입니다. 특히, 선구적인 연구 Campàs와 동료24 삽입 된 microdroplets를 사용 하 여 셀룰러 스트레스 척도를 새로운 방법을 개발. 그럼에도 불구 하 고,이 방법 세포 수준, 그러므로 힘에서 조직 또는 유기 체 수준 프로브 적용 되지 않습니다에서 세력을 측정을 수 있었습니다. 이 문서에 소개 된 프로토콜은 부분적으로이 갭을 채우기 위해 개발 되었다.

<table border="0" cellpadding="0" cellspacing="0" width="751">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Fertilized Specific pathogen-free White Leghorn chicken eggs</td>
			<td style="width:177px;">Charles River</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:288px;">Optical Coherent Tomography Microscope</td>
			<td style="width:177px;">Thorlabs</td>
			<td style="width:119px;">GAN220C1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:288px;">Silicone elastomer</td>
			<td style="width:177px;">Smooth-On, Inc.</td>
			<td style="width:119px;">EcoFlex 00-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Dissecting microscope</td>
			<td style="width:177px;">Leica</td>
			<td style="width:119px;">MZ8</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:288px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td style="width:177px;">Lonza</td>
			<td style="width:119px;">12-604F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Antibiotics</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">P4083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Chick serum</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">C5405</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Micropipette puller</td>
			<td style="width:177px;">Sutter Instrument</td>
			<td style="width:119px;">Model P-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Filter paper</td>
			<td style="width:177px;">Whatman</td>
			<td style="width:119px;">5202-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Phosphate buffered saline (PBS)</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">21-040-CV</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:288px;">Comsol MultiPhysics</td>
			<td style="width:177px;">Comsol</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">3D computer graphics software</td>
			<td style="width:177px;">Rhino 5</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Microscope attached with OCT</td>
			<td style="width:177px;">Nikon&#160;</td>
			<td style="width:119px;">FN1</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:288px;">Digital single-lens reflex camera</td>
			<td style="width:177px;">EOS&#160;</td>
			<td style="width:119px;">Rebel T3i</td>
			<td></td>
		</tr>
	</tbody>
</table>

probing the roles of physical forces in early chick embryonic morphogenesis

배아 개발은 전통적으로 바이오 유전자의 관점에서 하지만 morphogenesis에서 역학의 기본적인 중요성은 점점 더 인식 되 고. 특히,는 미 발달 병아리 마음과 뇌 관, 그들은 개발 형태학 상 변화를 받아야, 총리 후보자 morphogenesis 물리적 힘의 역할을 연구 중입니다. 진보적인 복 부 굽 힘과 관 미 발달 병아리 두뇌의 우 비틀림 기관 수준 좌우 비대칭 병아리 배아 개발에서의 초기 단계에서 발생. Vitelline 막 (VM) 태아의 등 쪽을 구속 하 고 개발 두뇌의 비틀림을 유도 하는 데 필요한 힘 제공에 연루 되어 있다. 여기 새로운 전 비켜 실험의 조합을 뇌 비틀림의 메커니즘을 식별 하기 위해 모델링 물리적 선물이. 햄버거-해밀턴 단계에서 11, 배아는 수확 하 고 교양 비켜 ex (미디어에서). VM 이후에 가져온된 모 세관 튜브를 사용 하 여 제거 됩니다. 액체의 레벨을 제어 하 고 액체 공기 인터페이스에 배아를 쓰는, VM의 기계적 역할을 대체 하는 미디어의 액체 표면 장력을 사용할 수 있습니다. 미세 실험 또한 뇌 비틀림의 카이랄성에 결과 변화를 찾을 수 심장의 위치 변경을 수행 했다. 이 프로토콜에서 결과 morphogenesis 운전 역학의 기본적인 역할을 설명 합니다.

이 연구에서는 HH11에서 배아의 VM 흉부 굴곡을 앞쪽 끝에서 제거 되었습니다. 배아 10 월 시스템에 의해 몇 군데 있었다. 이 단계에서 뇌 관의 비틀림 하지 (그림 1A)를 시작 했다. HH15-16에는 알을 품 되 고, 후 그들의 VM 제거와 배아 감소 뇌 튜브 비틀림, 약 35도 (그림 1B) 약 90도의 비틀림을 전시 하는 배아를 제어에 비해 전시. 미디...

Watch this Scientific Journal Video about Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis at JoVE.com

Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis

초기 여자 배아 Morphogenesis 물리적 힘의 역할을 프로 빙

여기, 우리는 일련의 새로운 전 비켜 실험 및 초기 여자 배아 뇌 비틀림 동안 morphogenesis의 역학을 공부에 대 한 물리적 모델링 접근법을 소개 하는 프로토콜을 제시.

Embryonic development is traditionally studied from the perspective of biomolecular genetics, but the fundamental importance of mechanics in morphogenesis ...

probing-roles-physical-forces-early-chick-embryonic

Research

JoVE Journal

Bioengineering

7.2K 조회수.  Dartmouth College. 여기, 우리는 일련의 새로운 전 비켜 실험 및 초기 여자 배아 뇌 비틀림 동안 morphogenesis의 역학을 공부에 대 한 물리적 모델링 접근법을 소개 하는 프로토콜을 제시.

비디오: Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Langendorff - perfused 토끼 하트의 Multiparametric 광 매핑

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

연구

생체공학

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

마우스 중심에 액션 후보와 칼슘 과도의 광학 매핑

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in these structures are held together by physical interactions mediated by a network of extracellular polymeric substances. Bacterial biofilms impact many human activities and the understanding of their properties is crucial for a better control of their development &#8212; maintenance or eradication &#8212; depending on their adverse or beneficial outcome. This paper describes a novel methodology aiming to measure in situ the local physical properties of the biofilm that had been, until now, examined only from a macroscopic and homogeneous material perspective. The experiment described here&#160;involves introducing magnetic particles into a growing biofilm to seed local probes that can be remotely actuated without disturbing the structural properties of the biofilm. Dedicated magnetic tweezers were developed to exert a defined force on each particle embedded in the biofilm. The setup is mounted on the stage of a microscope to enable the&#160;recording of time-lapse images of the particle-pulling period. The particle trajectories are then extracted from the pulling sequence and the local viscoelastic parameters are derived from each particle displacement curve, thereby providing the 3D-spatial distribution of the parameters. Gaining insights into the biofilm mechanical profile is essential from an engineer&#39;s point of view for biofilm control purposes but also from a fundamental perspective to clarify the relationship between the architectural properties and the specific biology of these structures.

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

This paper shows an original methodology based on the remote actuation of magnetic particles seeded in a bacterial biofilm and the development of dedicated magnetic tweezers to measure in situ the local mechanical properties of the complex living material built by micro-organisms at interfaces.

에 대한 마이크로 미터 프로브의 원격 자기 발동<em&gt; 현장에서</em세균 바이오 필름 물성&gt; 3D 매핑

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

매개 변수화 이완기 충전 형식주의를 통해 Transmitral 흐름의 운동 학적 모델링 기반의 분석에 의하여 세계적인 이완기 기능의 정량화

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

<em&gt; 체외</em쥐의 추간판의&gt; 장기 문화 모델

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

생체 외에서 Neuroinflammation의 모델링에 대 한 기본 Glial 세포의 3 차원 하이드로 겔 문화 개선

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

올레산의 Tripeptide 안정 Nanoemulsion

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

조직 수준 역학을 제어하기 위해 준수 기판에 인간 배아 줄기 세포 식민지의 기하학적 패터닝

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

조기 골관절염을 검출하기 위한 원자력 현미경 검사법 적용

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...

DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical for regulating many processes ranging from development to immunology. Therefore, developing the tools to study these forces at the molecular scale is critical. Our group developed a suite of molecular tension sensors to quantify and visualize the forces generated by cells and transmitted to specific ligands. The most sensitive class of molecular tension sensors are comprised of nucleic acid stem-loop hairpins. These sensors use fluorophore-quencher pairs to report on the mechanical extension and unfolding of DNA hairpins under force. One challenge with DNA hairpin tension sensors is that they are reversible with rapid hairpin refolding upon termination of the tension and thus transient forces are difficult to record. In this article, we describe the protocols for preparing DNA tension sensors that can be "locked" and prevented from refolding to enable "storing" of mechanical information. This allows for the recording of highly transient piconewton forces, which can be subsequently "erased" by the addition of complementary nucleic acids that remove the lock. This ability to toggle between real-time tension mapping and mechanical information storing reveals weak, short-lived, and less abundant forces, that are commonly employed by T cells as part of their immune functions.

This paper describes a detailed protocol for using DNA-based tension probes to image the receptor forces applied by immune cells. This approach can map receptor forces &gt;4.7pN in real-time and can integrate forces over time.

면역 세포에 의한 일시적인 피코네톤 수용체 힘을 매핑하는 DNA 장력 프로브

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical ...