1. 전 비켜 문화 프로토콜 : <ol><li> 수정된 닭 알을 품어 <ol class="lroman"><li> 수정된 닭고기 달걀은 13 저장할 수 있습니다 ° C를 부화하기 전에 오일을 개발을 시작하지 않고. 레드 와인 쿨러는 온도를 유지할 수 있습니다. </li><li> 72 시간 37.5에서 지속적인 락을 함께 60 %의 일정한 습도 보육 ° C에서 부화 계란은 뭉툭한 쪽을. </li></ol></li><li> 해먹스 (그림 1A)를 준비 <ol class="lroman"><li> 기입 어 9온스 플라스틱 컵의 따뜻한 물을 멸균. 일반 9온스 컵 8cm의 최고의 직경이 있습니다. 우리는이 정도 크기의 이곳 표시 오랫동안 문화 응용 프로그램에 가장 적합한 것으로 나타났습니다. </li><li> 컷 약 20 cm가 랩 집착. circumferentially 주변 고무 밴드를 배치하여 컵 위에 접사. 플라스틱은 물 위에 확산되어야합니다. 밴드 주변의 과도한 플라스틱 컷. 우리는 운송 중에 노른자 분리의 결과로, 노른자의 접착력을 방지 때문에이 응용 프로그램에 가장 적합하도록 포장을 집착 발견했습니다. </li><li> 70 % 에탄올로 침수 kimwipes (킴벌리 - 클락, 주식 회사)와 플라스틱을 닦아주십시오. </li></ol></li><li> 부화 후 72 시간 후 배양기에서 계란을 제거합니다. </li><li> 70 % 에탄올 침수 kimwipes로 표면을 닦는하여 달걀을 소독. </li><li> 배아의 적절한 위치 (그림 1B) 1-2 분 수평 알을 낳습. 계란 판지 상자는 가로로 누워있는 계란을 사용할 수 있습니다. 배아 1-2 분 후에 위로 회전합니다. </li><li> 층류 후드에서 무균 해먹스에 배아를 전송합니다. <ol class="lroman"><li> 계란 (그림 1C)의 아래쪽에 작은 덴트가있을 때까지 날카로운 가장자리 (양동이 또는 유리 비커의 가장자리와 같은)를 사용하여 부드럽게 계란을 누릅니다. 기억, 배아는 upperside에 위치합니다. </li><li> 자국의 반대편에 엄지손가락을 넣고 (그림 1D) 떨어져 수평 껍질을 가져옵니다. </li><li> 배아로뿐만 아니라 알부민 해먹 (그림 1E)에 노른자를 넣습니다. 알부민은 오랫동안 문화 관행에 대한 배아에 영양을 제공뿐만 아니라 시스템을 이동하여 유도 충격을 흡수하기 위해 노른자 주위 댐퍼닝 환경을 제공합니다. 해먹 아래 물 물 순환 히터를 사용하는 경우 배가 이미징 동안 운송 도중 땅에 병렬 머물면서 더위의 지휘자 역할을합니다 수 있도록합니다. </li><li> 전송이 제대로 수행되는 경우 배아가 이미 정상에 위치해야합니다. 그렇지 않다면, 확대해, 불임 개체 (닫힌 곡선 가위 ie. 블런트 끝)와 directionally 애무 배가 위로 회전 그러한 노른자를 사용합니다. </li><li> 배아를 봉인하기 위해 컵 위에 10cm 직경 페트리 접시를 놓습니다. </li></ol></li><li> 37.5에서 보육 세트로 컵 문화 플레이스 ° C. 휴대용 인큐베이터를 사용하는 경우, 증류수로 가득 9월 1일부터 2일까지 오즈의 마법사 &#39;컵을 배치하는 것은 ~에 습도를 유지 60% (그림 1 층). </li><li> 배아 일 7.2 이상 culturing의 배아는 조각 달걀 껍질 조각은 뼈 개발과 성숙을위한 칼슘 소스로 배아의 주변에 흩어져해야하는 경우. </li></ol> 외부 인큐베이터 영상 / 조작을위한 액세서리 장비 : 순환 물 목욕은 배양 온도를 유지하기 위해 집안에 지어진 동안 영상이나 조작 (그림 3). 컵 맞는 물 목욕은 물을 저수지에 맞는 크기의 튜브를 통해 연결되어 있습니다. 저항 히터는 물의 온도에 따라 수동으로 규제하는 저수지에서 물을 가열. 펌프가 물을 순환. Pictoral 표현 ultrasongraphy / florescent 현미경 (그림 3)를 통해 이미징 편리 모습. <img src="/files/ftp_upload/2154/2125fig1.jpg" alt="그림 1" /> 그림 1. 해먹 및 해먹스에 닭 배아의 양도의 준비. <img src="/files/ftp_upload/2154/2125fig2.jpg" alt="그림 2" /> 그림 2. 대표 결과 -. 예 비켜 문화 기술을 통해 재배 여러 단계 닭 배아 HH 17 이후 태아는이 기술을 통해 성장하실 수 있습니다. <img src="/files/ftp_upload/2154/2125fig3.jpg" alt="그림 3" /> 그림 3. 이상적인 개발 온도에서 배아를 유지하는 전 비켜 문화 시스템과 함께 사용하는 물 순환 시스템의 개략도 및 대표 사진. 2. 선택된 애플 리케이션 : I.의 Microinjection 이 배아 문화는 솔루션 대비 기반 이미징 (예 형광 현미경, 마이크로 계산된 지형 등) (그림 4) vasculature에 주입해야 microinjection 응용 프로그램에 적합합니다. 아래 microinjection 프로토콜은 다음과 같습니다 : <ol><li> 주사 appara 준비내밀어 <ol class="lroman"><li> 패션은 microforge를 사용하여 beveled 팁 microneedles에 0.75 ID 유리 모세관 튜브를 뽑아. 필요한 팁의 크기가 원하는 목표 정맥 직경 / 유동 속도에 따라 다릅니다. </li><li> 0.03 인치 ID의 실리콘 튜브에 주사기를 연결합니다. </li><li> 주입하는 솔루션으로 튜브를로드하고 튜브에 microneedle를 연결합니다. </li><li> 주사기 홀더 또는 주사기 펌프에 micromanipulator과 주사기에 microneedle를 놓습니다. </li></ol></li><li> 컵에 따라서 배아를 인상 멸균 물을 추가합니다. 우리는 혈관 (예 vitelline 선박)에 바늘의 적절한 침투를 위해, 수평 침투가 필요하다고 경험. 이것은 배 (胚)가 컵의 가장자리와 같은 비행기에 있어야합니다. <ol class="lroman"><li> 배아에서 플라스틱을 가지고있는 고무 밴드를 꺼내. </li><li> 부드럽게 플라스틱 한쪽을 들어 컵 일부 멸균 물을 추가합니다. </li><li> 다시 플라스틱에 고무 밴드를 고정합니다. </li></ol></li><li> 배아 선박에 대한 해결책을 주사. 우리의 경험에서 솔루션 vitelline 용기에 주입되었을 때, 출혈을 유발하지 않고 효율적인 재관류를 유지하기 위해 혈관 직경의 크기 약 10분의 1 일 있어야만 microneedles. <ol class="lroman"><li> 기포가 전혀 형성을 보장하지 microneedle에 대한 해결책을 펌프. </li><li> 선택한 그릇에 microneedle의 팁을 맞춥니다. vitelline 선박 들어, 포크 영역을 선택하면 선박에 침투 최대 영역을 제공합니다. </li><li> 배를 향에 바늘을 밀어. 배는 먼저 철회합니다. 바늘이 침투되면, 재관류를 시작합니다. 흐르는 혈액의 색깔은 재관류로 변경해야합니다. 색상의 변화가 본적이되지 않으면 바늘이 혈관 밖으로 침투 수 있습니다. 선박의 색상 변경을 볼 때까지 동시에 솔루션을 양수하면서 그 경우에는 천천히 microneedle을 철회. 재관류가 완료되면, 선박의 microneedle을 철회. </li></ol></li></ol><img src="/files/ftp_upload/2154/2125fig4.jpg" alt="그림 4" /> 그림 4 : 전 비켜 교양 여자는 배아의 vasculature에 형광 염료의 Microinjection. II. Microsurgical 절차 - 왼쪽 심방세동 내고 이 전 비켜 문화 설치는 배아에 대한 모든 액세스를 제공 이후 여자는 태아에 외과 응용 프로그램을 수행하는 데 이상적입니다. 아래 예제 기술에 대한 프로토콜은, 심방 내고 (랄) (그림 5)은 떠났다. 절차는 약 24 시간 문화 시대 (HH24)로 수행됩니다 : <ol><li> 매듭을 overhand 준비 ~ 10-0 나일론 수술 봉합사를 사용하여 0.5 mm의 직경. </li><li> 고급 집게로 로컬 배아 통해 chorionic와 allantoic 세포막을 제거하고 배아의 왼쪽 볼 것을 그것이 수직으로 같은 회전 뒷부분에서 배아를 리프트. </li><li> 고급 집게를 사용하여 좌심방을 통해 심낭을 제거합니다. </li><li> 좌심방을 통해 매듭을 정렬하고 조입니다. 왼쪽에 원래의 혈액 흐름 ~ 75 % 금지로 매듭 너무 묶여 있어야합니다. microscissors와 매듭의 가장자리를 잘라 조심스럽게 초과 봉합사를 제거하고 폐기. </li><li> 원래 방향 (위에서 오른쪽)에 다시 배아를 회전합니다. </li><li> 섐 컨트롤은 동일한 절차를 포함되지만 봉합사를 막 통과하고 묶여되지 않습니다. 효과 랄 치료 (75 % 수축)가 24시간 수술에서 확인되며, 자격이 배아는 제외됩니다. </li></ol><img src="/files/ftp_upload/2154/2154fig5.jpg" alt="그림 5" /> 그림 5 :. 컨트롤과 왼쪽 심방 출혈도 잡았 여자의 배아 화살표는 출혈도 잡았 배아의 75 퍼센트 작은 왼쪽 심방을 보여줍니다.

<ol>
	<li>Zamir, E. A., Czirok, A., Cui, C., Little, C. D., Rongish, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesodermal+cell+displacements+during+avian+gastrulation+are+due+to+both+individual+cell-autonomous+and+convective+tissue+movements.">Mesodermal cell displacements during avian gastrulation are due to both individual cell-autonomous and convective tissue movements.</a> Proc Natl Acad Sci U S A. 103, 19806-19811 (1980).</li><li>Ehrman, L. A., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+regulation+in+the+heart+forming+region+of+avian+embryos.">Lack of regulation in the heart forming region of avian embryos.</a> Dev Biol. 207, 163-175 (1999).</li><li>Clark, E. B., Hu, N., Rosenquist, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+conotruncal+constriction+on+aortic-mitral+valve+continuity+in+the+stage+18,+21+and+24+chick+embryo.">Effect of conotruncal constriction on aortic-mitral valve continuity in the stage 18, 21 and 24 chick embryo.</a> Am J Cardiol. 53, 324-327 (1984).</li><li>deAlmeida, A., McQuinn, T., Sedmera, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+ventricular+preload+is+compensated+by+myocyte+proliferation+in+normal+and+hypoplastic+fetal+chick+left+ventricle.">Increased ventricular preload is compensated by myocyte proliferation in normal and hypoplastic fetal chick left ventricle.</a> Circ Res. 100, 1363-1370 (2007).</li><li>Nakamura, H., Funahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+DNA+into+chick+embryos+by+in+ovo+electroporation.">Introduction of DNA into chick embryos by in ovo electroporation.</a> Methods. 24, 43-48 (2001).</li><li>Butcher, J. T., McQuinn, T. C., Sedmera, D., Turner, D., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transitions+in+early+embryonic+atrioventricular+valvular+function+correspond+with+changes+in+cushion+biomechanics+that+are+predictable+by+tissue+composition.">Transitions in early embryonic atrioventricular valvular function correspond with changes in cushion biomechanics that are predictable by tissue composition.</a> Circ Res. 100, 1503-1511 (2007).</li><li>McQuinn, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+ultrasonographic+imaging+of+avian+cardiovascular+development.">High-frequency ultrasonographic imaging of avian cardiovascular development.</a> Dev. Dyn. 236, 3503-3513 (2007).</li><li>Korn, M. J., Cramer, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Windowing+chicken+eggs+for+developmental+studies.">Windowing chicken eggs for developmental studies.</a> J Vis Exp. , (2007).</li><li>Auerbach, R., Kubai, L., Knighton, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+procedure+for+the+long-term+cultivation+of+chicken+embryos.">A simple procedure for the long-term cultivation of chicken embryos.</a> Dev Biol. 41, 391-394 (1974).</li></ol>

관심 없음 충돌 선언하지 않습니다.

광 접속 및 조류 배아의 실험은 알 껍질의 제약으로 인해 도전이다. 윈도우 크게 분사하고 microsurgical 접근 8 액세스할 수 microvessels의 수를 제한합니다. 결과적으로 초기 단 배아는 조작과 지속적인 관찰이 불가능하실 수 있습니다. 장기 문화 9 방해 배아에서 표면 장력의 불충 분한 제어하기 때문에 배양 접시를 사용하여 초기 전 비켜 문화는 제한된 사용했다. 우리는 최근에 허용 표면 tenstion 7 유지 육각형 무게는 보트를 사용하여이 기술을 개선, 이러한 문화는 전송하기 어려웠다. 여기에 제공 기술은이 문제를 해결하고 크게 microinjection과 수술 절차를 포함하여 고용 수있는 실험 방법과 영상 기술의 종류를 확장합니다. 연구 나중에 morphogenic 이벤트를 이해에 초점을 맞추고,이 기술은 모니터링 및 현재 실험적으로 지속적인 연구 개발 불가능 이벤트의 변조를 사용합니다.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Fertile white Leghorn chicken eggs</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Model GB1, Avery Incubators, Hugo CO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Saran Wrap</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Kimwipes</td>
<td>&nbsp;</td>
<td>Kimberly-Clark, Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Rubber bands</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Warm sterile water</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>9 oz plastic cup</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>100 mm diameter Petri dish</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1602N thermal air</td>
<td>&nbsp;</td>
<td>GQF Manufacturing CO, Savannah GA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescein-conjugated dextran (2 MDa, 1% w/v in phosphate buffered saline)</td>
<td>&nbsp;</td>
<td>Sigma Aldrich Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microforge</td>
<td>&nbsp;</td>
<td>Glassworx, Inc, St Louis MO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glass capillary tubes (0.75 mm ID)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulator</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Sarasota FL</td>
<td>Model M3301L</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent microscopy</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>Z20</td>
<td>&nbsp;</td>
<tr>
<td>Fine 55-forceps</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Sarasota FL</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10-0 nylon surgical suture</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tubing</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>1mm OD</td>
</tr>
<tr>
<td>3 mL syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>200 &mu;L pipetter and pipette tips</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

an ex-ovo chicken embryo culture system suitable for imaging and microsurgery applications

정상 및 기형 배아 개발을 운전 유전과 microenvironmental 요인 사이의 관계를 이해하는 것은 새로운 치료 전략을 발견하기위한 필수적입니다. 이미징 기술의 발전은 조직과 신체 계획의 성숙의 양적 조사를 활성화하지만, 나중에 무대 배아 morphogenesis 덜 분명하다. 치킨 배아 때문에 문화와 수술 조작의 용이성이 응용 프로그램에 대한 매력적인 척추 동물 모델 시스템입니다. 초기 배아는 세포 patterning과 운명을 연구 1,2을위한 완벽한 광 액세스를 가능하게 필터 종이 링에 짧은 시간 동안 교양 수 있습니다. 같은 심장 morphogenesis로 유학 고급 개발 프로세스는 전통적으로 달걀 껍질 3-5의 창문을 통해 수행하지만,이 기술은 창 크기에 따라 광학 접근을 제한하고 있습니다. 최대 초음파 6,7 통해 고해상도 이미징을 활성화 10 일까지 우리는 이전에 육각 무게 보트에서 전 비켜 문화 전체 배아를 간단한 방법을 개발했습니다. 이러한 문화는 라이브 실험에 사용할 수 이미징 도구의 유형을 제한, 운송하기 어려웠다. 우리는 여기서 비용 효율적인 휴대용 환경 챔버와 개선된 쉘 적은 문화 시스템을 제시한다. 계란은 부분적으로 살균 물로 채워진 플라스틱 컵에 circumferentially 부착된 폴리 우레탄 막 (포장을 집착)에 의해 만들어진 그물에 금이 있었다. 아래 해먹와 물의 역학 교통으로 유도 진동을 저해할 도움 동안 해먹의 원주와 깊이의 크기는 두 표면 장력을 유지하기 위해 중요했다. 물 목욕을 순환 작은 풋프린트 또한 실험 기간 동안 지속적인 온도 제어를 사용하도록 개발되었습니다. 우리는 morphogenic 결함없이 적어도 14 일 동안 이런 방식으로 문화 배아 수있는 능력을 입증 또는 여러 microsurgical 및 이미징 응용 프로그램에서이 시스템을 지연하고 사용합니다.

Watch this Scientific Journal Video about An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications at JoVE.com

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

이 문서에서는, 우리는 장기 사용하는 간단한 방법을 제시<em&gt; 전 비켜</em&gt; 조류 배아의 문화. 이 기술은 전체 광 액세스 및 / 또는 조류 배아의 살균 운송을 필요로하는 길이 실험​​에 이상적입니다.

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

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

an-ex-ovo-chicken-embryo-culture-system-suitable-for-imaging

Nanyang Technological University

Research

JoVE Journal

Biology

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

22.9K 조회수.  Cornell University. 이 문서에서는, 우리는 장기 사용하는 간단한 방법을 제시 전 비켜 조류 배아의 문화. 이 기술은 전체 광 액세스 및 / 또는 조류 배아의 살균 운송을 필요로하는 길이 실험​​에 이상적입니다.

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

Windowing Chicken Eggs for Developmental Studies

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling.  In addition, it allows for molecular perturbations of several types, including placement of protein-coated beads and introduction of plasmid DNA using in ovo electroporation.  These experiments have yielded important data on the development of the central and peripheral nervous systems.  For many of these studies, it is necessary to open the eggshell and reclose it without perturbing the embryo's growth.  The embryo can be examined at successive developmental stages by re-opening the eggshell.  While there are several excellent methods for opening chicken eggs, in this article we demonstrate one method that has been optimized for long survival times.  In this method, the egg rests on its side and a small window is cut in the shell.  After the experimental procedure, the shell is used to cover the egg for the duration of its development.  Clear plastic tape overlying the eggshell protects the embryo and helps retain hydration during the remainder of the incubation period.  This method has been used beginning at two days of incubation and has allowed survival through mature embryonic ages.

The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling. In this article we demonstrate one egg preparation method that has been optimized for long survival times.

발달 연구를위한 윈도우 치킨 계란

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has ...

연구

생물학

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform routine activities, but prosthetic suspensions using traditional socket technology can prove to be cumbersome and uncomfortable for a person with limb loss.  Moreover, for those with high proximal amputations, limited residual limb length may prevent exoprosthesis attachment all together. Osseointegrated implant technology is a novel operative procedure which allows firm skeletal attachment between the host bone and an implant. Preliminary results in European amputees with osseointegrated implants have shown improved clinical outcomes by allowing direct transfer of loads to the bone-implant interface.  Despite the apparent advantages of osseointegration over socket technology, the current rehabilitation procedures require long periods of restrictive load bearing prior which may be reduced with expedited skeletal attachment via electrical stimulation.  The goal of the osseointegrated intelligent implant design (OIID) system is to make the implant part of an electrical system to accelerate skeletal attachment and help prevent periprosthetic infection. To determine optimal electrode size and placement, we initiated proof of concept with computational modeling of the electric fields and current densities that arise during electrical stimulation of amputee residual limbs.  In order to provide insure patient safety, subjects with retrospective computed tomography scans were selected and three dimensional reconstructions were created using customized software programs to ensure anatomical accuracy (Seg3D and SCIRun) in an IRB and HIPAA approved study.  These software packages supported the development of patient specific models and allowed for interactive manipulation of electrode position and size. Preliminary results indicate that electric fields and current densities can be generated at the implant interface to achieve the homogenous electric field distributions   required to induce osteoblast migration, enhance skeletal fixation and may help prevent periprosthetic infections.  Based on the electrode configurations experimented with in the model, an external two band configuration will be advocated in the future.

There is a need to develop alternative prosthesis attachment due to limb loss attributed to vascular occlusive diseases and trauma. The goal of the work is to introduce an osseointegrated intelligent implant design system to increase skeletal fixation and reduce periprosthetic infection rates for patients needing osseointegrated technology.

절단 수술을받은 사람에 대한 Osseointegrated 지능형 임플란트 설계 시스템의 생체 분석

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform ...

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

병아리 예 비켜 문화와 예 비켜 CAM 어세이 : 그게 정말로 작동하는 방법

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

치킨의 배아에 인간 줄기 세포의 Xenotransplantation

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

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

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

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

회전 형 바틀 문화 시스템을 사용 로던트 전체 엠브료 문화의 방법

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

Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice. HYPERStack vessels allow closed system scale up from the current stacked plate products and delivers >2.5X more cells in the same volumetric footprint. The HYPERStack vessels function via gas permeable material which allows gas exchange to occur, therefore eliminating the need for internal headspace within a vessel. The elimination of headspace allows the compartment where cell growth occurs to be minimized to reduce space, allowing more layers of cell growth surface area within the same volumetric footprint.
For many applications such as cell therapy or vaccine production, a closed system is required for cell growth and harvesting. The HYPERStack vessel allows cell and reagent addition and removal via tubing from media bags or other methods.
This protocol will explain the technology behind the gas permeable material used in the HYPERStack vessels, gas diffusion results to meet the metabolic needs of cells, closed system cell growth protocols, and various harvesting methods.

An Introduction into the technology, protocol and handling of the Corning HYPERStack Vessels and accessories used for high yield adherent cell culture. The protocol will show how to use the closed system vessels for increasing cell harvesting over current stacked plate products.

가스 투과 재료 기술로 HYPERStack 용기를 사용하여 닫힌 체계의 세포 배양 프로토콜

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice.   ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

에 의한 이전 치킨 태아에 유전자 이체<em&gt; 전 비켜</em&gt; Electroporation

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

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 ...

An Ex vivo Culture System to Study Thyroid Development

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

An Ex vivo Culture System to Study Thyroid Development

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

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

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

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

전 비켜 치킨의 배아 문화 시스템