Name: primary endodermal epithelial cell culture from the yolk sac membrane of japanese quail embryos
Uploaded: 2016-03-10T13:00:00.000+00:00
Duration: 11 min 53 s
Description: Watch this Scientific Journal Video about Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos at JoVE.com

현재 프로토콜의 개발을위한 자금 조달은 특히 국립 대만 대학, 대만 (부여 ID-104R350144)뿐만 아니라 대만의 과학 기술부 (부여 ID &quot;상위 대학 계획에 대한 목표&quot;에 의해 지원됩니다 MOST (104) -2313-B-002-039-MY3). 우리는 특히 현재의 연구를위한 동물 실 및 실험실 공간을 제공하기 위해 국립 대만 대학의 생명 공학 센터 감사합니다.

참고 :..이 절차는 등 바우어 등 2013 나카자와 등, 2011 년 2,9에 의해 개발 된 치킨 모델 문화 프로토콜의 수정입니다 1. 건강한 배아 5 일 태아 일본어 메추라기에서 준비 <ol><li> 같은 케이지에서 함께 한 남자와 세 여성의 성적으로 성숙한 일본어 메추라기를 놓습니다. 전원 공급 및 임의 량의 물. 빛의 14 시간과 어둠의 10 시간에 동물 방에 빛을 조정합니다. </li><li> 오후에 매일 비닐 봉지에 수정 된 알을 수집하고 태아의 성장 속도를 지연하기 위해 16 ° C 냉장고에 보관하십시오. </li><li> 2 주 기간 내에 계란의 충분한 수를 수집 한 후, 5 일 동안 통풍이 잘되는 37 ° C 배양기에서 수정 된 알을 품어. </li></ol> 2. 기초 배지 및 세척 솔루션을 준비 <ol><li> 성장 배지로 (pH를 7.2로 조정) DMEM / F-12 배지를 사용하고, 그것을 보충10 % 새로 태어난 송아지 혈청 (NBCS), 1 % 펜 연쇄상 구균 Ampho와. 솔루션 (PSA; 페니실린, 105 단위 / L, 스트렙토 마이신, 100 ㎎ / ℓ, 암포 테리 신 B, 0.25 ㎎ / ℓ). </li><li> 저장을위한 10 배 농도의 인산 완충 식염수 (PBS) 용액을 제조; 씻어 (pH를 7.2로 조정;의 KCl, 2.667 밀리미터, KH 2 PO 4, 1.471 밀리미터; 나 2 HPO 4 -7H- 피 2 O, 8.06 mM의 NaCl을, 137.93 밀리미터 포함)과 난황 막 동안 축축하게 1X PBS 솔루션을 사용 계란 노른자와 알부민에서 내배엽을 분리. </li><li> 내배엽 펠렛을 다시 중단 (pH를 7.2로 조정) DMEM 배지를 사용합니다. </li></ol> 일본어 메추라기 YSMs에서 차 EECS 3. 컬렉션 <ol><li> 오일 (단계 1.3)에 대한 수집 한 알을 품어. 정상 배아 발달을 확인 달걀 ​​캔 들러에 의해 계란을 검사합니다. 참고 : 배아 일 5 일 YSM의 중배엽의 경계는 매우 명확하고 3 배아 층은 덜 함께 밀접하게 연결되어있다, 그래서 OB하기 쉬운주석 박의 내배엽. 만 YSM 컬렉션 정상적인 모세 혈관 순환 개발을 보여주는 계란을 사용합니다. </li><li> 주의 깊게 신선한 물과 75 % 에탄올을 사용하여 껍질을 청소합니다. 부드럽게, 가위로 공기 셀 위치에서 달걀 껍질을 열고 밖으로 껍질, 37 ° C 1X PBS로 가득 10cm 배양 접시에 집게와 장소에 의해 전체 YSM를 수집합니다. </li><li> 가위는 배아, 난황과 난백을 분리하고 부드럽게 1X PBS로 세척 YSM 세 번 및 10cm 배양 접시에 PBS 용액 YSM 배치하여. </li><li> YSM에서 외배엽을 제거하기 위해 집게와 가위를 사용합니다. 집게를 사용하여 2 번 단단히 내배엽 세포 층을 보유하고, 다른 하나는 모세관 중배엽을 잡아. </li><li> 해부 현미경에서 떨어져 당겨 배아를 향한 방향으로 중배엽의 가장자리에서 내배엽를 구분합니다. 37 ° C 와트 성장 배지 15 ㎖ 중의 적기 장소를 이용하여, 50 ml 원심 분리 튜브 내에서 담황색 내배엽 모아서어 목욕 모든 조직 모음이 완료 될 때까지. </li></ol> 콜라게나 소화에 의해 내배엽 조각 4. 소화 <ol><li> 갓 DMEM 10 ml의 유형 IV 콜라​​게나 6.5 단위를 녹인다. </li><li> RT에서 3 분 동안 130 XG에 50 ㎖ 튜브 (3.4)을 원심 분리기. 매체를 기음과 피펫을 사용하여 6cm 배양 접시에 모든 내배엽을 배치합니다. 이 배양 접시에 1 ml의 콜라게나 솔루션을 추가합니다. </li><li> 각 조각의 크기까지 곡선 가위 2 메추라기 배아에서 수집 내배엽 슬라이스 대략 2-3mm이다. , 적기를 사용하여 신선한 콜라게나 솔루션의 9​​ ml의와 함께 50 ㎖ 원심 분리 튜브에 모든 조각을 수집합니다. </li><li> 175 rpm에서 37 ° C 진탕 수조에서 30 분 동안 콜라게나 조직 다이제스트. 콜라겐이 부분 소화 이식편의 세포 증식을 향상시키는 데 사용된다. </li><li> RT에서 3 분 동안 130 XG에 원심 분리기 부드럽게 피펫을 사용하여 상층 액의 일부를 제거. 20 ㎖ DMEM에 현탁하여 펠렛을 씻고 3 분 130 XG에 원심 분리 후 상층 부분을 제거합니다. </li><li> 다시 중단 성장 매체 (10 % NBCS 1 %의 PSA와 DMEM / F12)의 12 ml의와 펠렛을, 24 웰 플레이트에 부드럽게와 동일 (각 웰에 500 ㎕의 세포 솔루션을) 시드. </li><li> 세포 조직에서 증식 할 수 있도록 2 일 동안 공기 중에서 5 %에서 37 ° C에서 CO 2 조직 이식편 부화. </li><li> 추가 2 일 동안 세포를 인큐베이션하고 세포 생존 분석 프로토콜에 의해 세포 생존율에 대한 특성화. 15 주 : 살아있는 세포는 세포질 내에서 환원 환경을 유지한다. 레사 주린, 활성 성분은 푸른 색과 거의 비 형광 비 독성 세포 투과성 화합물이다. 세포 배양에 첨가 될 때, 레사 주린은 미토콘드리아 효소에 의해 세포질로 레조 루핀 (resorufin)로 감소된다. 레조 루핀 (resorufin)는 붉은 색과 높은 형광입니다. 실행 가능한 세포가 지속적으로 레사 주린은 레조 루핀 (resorufin)로 변환, incre주변 세포 배양 배지의 전체 형광 색 asing. </li><li> 한 바와 같이 전사 및 PCR 마커 유전자의 mRNA 발현의 정량적 실시간 역 내배엽 상피 세포의 총 RNA 분리를 수행한다. (16, 17)을 <ol><li> SOAT1 mRNA를 검출하는 역전사 효소를 사용하여 PCR (센스 : 5&#39;-GAAGGGGCCTATCTGGAACG-3 &#39;안티센스 : 5&#39;-ATCTGCACGTGACATGACCA-3&#39;; PCR 생성물을 B​​P = 168) EEC 세포 특이 마커로. 하우스 키핑 유전자 및 내부 통제로 β-굴지의 mRNA (PCR 제품 = 151 bp의 : &#39;; 5&#39; GTGATGGACTCTGGTGATGG-3 안티센스&#39;5&#39;- TGGTGAAGCTGTAGCCTCTC-3 감각)를 사용합니다. </li><li> RT-qPCR에 제품의 크기를 검출하는 겔 전기 영동을 사용한다. 1 % 아가 로스 젤을 준비, 2 μL 로딩 염료 10 μl의 qPCR에 제품을 혼합한다. 로드 10 μL 웰을 1 % 아가 로스 겔에서 각 혼합물에, 30 분 동안 100V로 겔을 실행. </li></ol></li></ol>

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There is no competing financial interests or other conflicts of interest. We did not get any funding from any private company.

전 배양 시스템은 제한된 성공을 갖기 때문에, 더 배양 시스템이 필요하다. 일본 메추라기 YSM의 내배엽은 전 생체 내 외식의 더 나은 성장 성능을 달성하기 위해 세포 - 세포 접합을 완화하기 위해 콜라겐 등의 단백질 분해 효소와 치료가 필요합니다. 우리의 데이터는 부분 소화 처리에서 세포 수는 이일 (그림 1)에 대한 시딩 후 소화되지 않은 조직 문화에서보다 더 컸다 것으로 나타?...

조류 배아의 주요 영양 자원이 33 % 지질, 17 %의 단백질로 구성 노른자, 1 % 재. 1 배아 개발 중입니다, 난황 막 (YSM)는 배아 복강 내에서 성장하고 점차적으로 노른자를 커버 표면. 배아 2 일에 시작하여, 지질 대사 및 혈관 신생과 관련된 유전자의 발현이 점차 YSM 증가되고 YSM 천천히 융모와 같은 돌기를 발생한다. -8,9- 이러한 돌기는 배아 발달을 지원하기 노른자 영양소의 흡수를 증가시킨다. YSM 세 가지 배엽, 내배엽, 중배엽과 외배엽을 포함하는 배외 (extraembryonic) 조직이다. (14) 난황 외배엽 부드럽게 난황을 포함하는 난황 막과 알부민 및 링크에 직면 해있다. 내배엽 상피 세포는 shap로 나눌 수있다 달걀 노른자를 향해 직접 직면하고 영양 활용 포털의 역할을한다. 6 YSM이 확장하면서, 내배엽 상피 세포 (EECS)를하고 있습니다두 그룹, 지역 난황 및 지역 vasculosa에 전자 및 기능을 제공합니다. (7) 영역 난황는 내배엽 세포로 구성되며 배아로부터 떨어진되고 영역 vasculosa는 중배엽 세포로 구성되고, 혈관 결합 조직으로 분화 EECS을 덮는다. 배아 5 일까지, 노른자는 완전히 YSM의 외배엽과 내배엽이 적용되고, 혈관 영역은 빠른 속도로 성장하고있다. YSM은 배아 순환 시스템으로 (노른자 유래 매우 저밀도 지질 단백질 등)과 단백질. 9, 2 따라서, 우리는 지질의 메커니즘을 연구하기 위해, 기본 일본어 메추라기 배아 내배엽 상피 세포 배양 시스템을 구축 recomposes 해제 지질 흡수 조류 배아 개발하는 동안 YSM에 활용. 예컨대 중성 지방, 레시틴, 인지질 및 콜레스테롤 에스테르 (CE)와 같은 지질 조류 배아의 주 에너지 원이다. 개발의 초기 단계에서, 난황 지질 C는1.3 %의 CE의 omposed하고 조류 배아 발달 (3)의 중간 10-15 %로 상승, 11. 콜레스테롤 에스테르 조류 배아 YSM에 스테롤 O-아실 트랜스퍼 라제 1 (SOAT1)에 의해 콜레스테롤로부터 합성된다. (4) 콜레스테롤의 저장 형태는 CE, CE는 지질 단백질에 실시하고, 지질 단백질은 조직에 혈액 순환에 의해 수송된다. 13 주 해치 전에 조류 배아의 급속한 성장이있다. 약 노른자의 나머지 지질 함량의 68 %는이 단계에서 흡수된다. 10 지질 EEC 연구 모델로 정화 할 수있다 이용된다 노른자하는 메커니즘. 닭 EEC 배양 프로토콜은 본 연구의 목표를 ​​달성하기 위해 설립되었다. 2, 9, 그러나, 향상된 EEC 세포 배양 방법에 의해 영양분 활용도를 매개 특정 효소와 단백질의 기능을 연구하는데 필요한 조직 이식편의 낮은 성공률로 개발하는 동안 조류 배아.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Gibco by Life technologies</td><td>12800-017 10 X 1 L</td><td>For wash the EECs pellets</td></tr><tr><td>D-MEM/F-12</td><td>Gibco by Life technologies</td><td>12400-024 10 X 1 L</td><td>As the basal medium in culturing EECs</td></tr><tr><td>NBCS</td><td>Gibco by Life technologies</td><td>16010-159</td><td>As the supplyment serum in culturing EECs</td></tr><tr><td>Pen-Strep Ampho. Solution</td><td>BI (Biological Industries)</td><td>03-033-1B 100 ml</td><td>For attenuating the possible infection&amp;nbsp;</td></tr><tr><td>Collagenase Type IV</td><td>Gibco by Life technologies</td><td>17104-019 1 g</td><td>&amp;nbsp;Collagenase is a protease with specificity for the bond between a neutral amino acid (X) and glycine in the sequence Pro-XGly-Pro. As the protease for dissociation of cells from primary tissue.</td></tr><tr><td>24-well plate</td><td>FALCON&lt;sup&gt;&amp;reg;&lt;/sup&gt;</td><td>REF-353047</td><td>For EECs to attach and extension</td></tr><tr><td>50 ml PP centrifuge tubes</td><td>Corning&lt;sup&gt;&amp;reg;&lt;/sup&gt; CentriStar&lt;sup&gt;TM&lt;/sup&gt;</td><td>430829</td><td>For transportion of membranes and enzyme digestion</td></tr><tr><td>50 ml Conical bottomed Tube with Cap</td><td>PRO TECH</td><td>CT-50-PL-TW</td><td>For transportion of membranes and enzyme digestion</td></tr><tr><td>Reciprocal shaking bath</td><td>DEAGLE</td><td>SB302</td><td>For better enzymatic digestion on membranes</td></tr></tbody></table>

primary endodermal epithelial cell culture from the yolk sac membrane of japanese quail embryos

우리는 개발 과정에서 조류 배아에 의해 영양소 이용률을 매개하는 특정 효소와 단백질의 기능을 연구하는 내배엽 상피 세포 배양 모델 (EEC)를 설립했다. 수정 된 일본어 메추라기 계란 5 일 동안 37 ° C에서 배양 한 다음 골목 막 (YSM)는 EEC 문화 시스템을 구축하기 위해 수집 노른자 하였다. 우리 YSM에서 배아 내배엽 층을 단리하고, (2) 내에 막 분리 - MM 3 개의 부분적으로 24 웰 배양 플레이트에 파종 전에게나로 분해. EECS는 조직에서 증식 및 세포 배양 연구를위한 준비가되어 있습니다. 우리는 EECS 예를 들어 생체 내에서 YSM의 일반적인 특성, 지질 방울의 축적, 스테롤 O-아실 트란스 퍼과 지단백 리파제의 발현을 한 것으로 나타났습니다. 부분 소화 치료는 크게 EEC 문화의 성공적인 속도를 증가했다. EECS을 활용, 우리는 SOAT1의 발현이 캠프 D에 의해 규제 것을 증명ependent 단백질은 관련 경로를 키나아제. 이 차 일본어 메추라기 EEC 문화 시스템은 배아 지질 수송을 연구하고 조류 배아 개발하는 동안 YSM 영양소 이용률을 매개에 관여하는 유전자의 역할을 명확히하기 위해 유용한 도구입니다.

일관성 있고 유용한 셀 모델을 설정하는 목표를 달성하기 위해, 우리는 확장 증식 속도 및 조류 EECS의 성능을 안정화 할 필요가있다. 우리는 부분적 콜라겐 또는 콜라겐 플러스 디스 파제 0.6 U와 같은 단백질 분해 효소로 분해 내배엽 아니 효소 소화와 내배엽의 직접 배양을 비교 하​​였다. 디스 파제는 비극성 아미노산 잔기의 N 말단 펩티드 결합을 가수 분해 아미노 - ?...

Watch this Scientific Journal Video about Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos at JoVE.com

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

일본 메추라기 배아의 노 른 골목 막에서 차 내배엽 상피 세포 배양

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient ...

primary-endodermal-epithelial-cell-culture-from-yolk-sac-membrane

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

8.8K 조회수.  National Taiwan University. To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

비디오: Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

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

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

Method for Culture of Early Chick Embryos ex vivo New Culture

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

조기 칙 태아 예 생체내 (새로운 문화)의 문화에 대한 방법

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

연구

생물학

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

크로스 종 이식에 의한 신경 크레스트 마이 그 레이션과 차별화 분석

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

신경과학

Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the formation of quail-chicken chimeras and the use of the chorioallantoic membrane (CAM) to sustain the development of ectopic tissues date back to the last century. Nowadays, the combination of these classical techniques with recent in vitro methodologies offers novel prospects to further explore organ formation.
Here we describe a two-step approach to study early- and late-stages of organogenesis. Briefly, the embryonic region containing the presumptive territory of the organ is isolated from quail embryos and grown in vitro in an organotypic system (up to 48 h). Cultured tissues are subsequently grafted onto the CAM of a chicken embryo. After 10 days of in ovo development, fully formed organs are obtained from grafted tissues. This method also allows the modulation of signaling pathways by the regular administration of pharmacological agents and tissue genetic manipulation throughout in vitro and in ovo developmental steps. Additionally, developing tissues can be collected at any time-window to analyze their gene-expression profile (using quantitative PCR (qPCR), microarrays, etc.) and morphology (assessed with conventional histology and immunochemistry).
The described experimental procedure can be used as a tool to follow organ formation outside the avian embryo, from the early stages of organogenesis to fully formed and functional organs.

This article provides an updated approach to the classical quail-chicken chimera system to study organ formation, by combining novel in vitro and in ovo experimental procedures.

2 단계 접근-그리고 늦은-초기 조류 모델에서 기관 형성의 탐구: Thymus 및 부 갑 상선 동맥 Organogenesis 패러다임

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the ...

발생학

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

Organotypic 문화 메서드 배아 치킨 조직 개발 연구를

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Quail Chorioallantoic Membrane - A Tool for Photodynamic Diagnosis and Therapy

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties make it an excellent in vivo experimental model to study angiogenesis, tumor growth, drug delivery systems, or photodynamic diagnosis (PDD) and photodynamic therapy (PDT). At the same time, this model addresses the requirement for the replacement of experimental animals with a suitable alternative. Ex ovo cultivated embryo allows easy substance application, access, monitoring, and documentation. The most frequently used is chick CAM; however, this article describes the advantages of the Japanese quail CAM as a low-cost and high-throughput model. Another advantage is the shorter embryonic development, which allows higher experimental turnover. The suitability of quail CAM for PDD and PDT of cancer and microbial infections is explored here. As an example, the use of the photosensitizer hypericin in combination with lipoproteins or nanoparticles as a delivery system is described. The damage score from images in white light and changes in fluorescence intensity of the CAM tissue under violet light (405 nm) was determined, together with analysis of histological sections. The quail CAM clearly showed the effect of PDT on the vasculature and tissue. Moreover, changes like capillary hemorrhage, thrombosis, lysis of small vessels, and bleeding of larger vessels could be observed. Japanese quail CAM is a promising in vivo model for photodynamic diagnosis and therapy research, with applications in studies of tumor angiogenesis, as well as antivascular and antimicrobial therapy.

The chorioallantoic membrane (CAM) of the avian embryo is a very useful and applicable tool for various areas of research. A special ex ovo model of Japanese quail CAM is suitable for photodynamic treatment investigation.

메추라기 융모막 막 - 광 역학 진단 및 치료를위한 도구

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties ...

암 연구학

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed contributions to unveiling key processes in developmental biology.
Herein is described an optimized method to isolate embryonic tissues from quail and chickens by microsurgery and enzymatic digestion while preserving its biological properties. After isolation, tissues from both species are associated in an in vitro organotypic assay for 48 h. Quail and chicken tissues can be discriminated by distinct nuclear features and molecular markers allowing the study of the cellular cross-talk between heterospecific association of tissues. This approach is, therefore, a useful tool for studying complex tissue interactions in developmental processes with highly dynamic spatial modifications, such as those occurring during pharyngeal morphogenesis and the formation of the foregut endoderm-derived organs. This experimental approach was first developed to study the epithelial-mesenchymal interactions during early-stages of thymus formation. In this, the endoderm-derived prospective thymic rudiment and mesoderm-derived mesenchyme, were isolated from quail and chicken embryos, respectively.
The capacity of the associated tissues to generate organs can be further tested by grafting them onto the chorioallantoic membrane (CAM) of a chicken embryo. The CAM provides nutrients and allows gas exchanges to the explanted tissues. After 10 days of in ovo development, the chimeric organs can be analyzed in the harvested explants by conventional morphological methods. This procedure also allows studying tissue-specific contributions during organ formation, from its initial development (in vitro development) to the final stages of organogenesis (in ovo development).
Finally, the improved isolation method also provides three-dimensionally (3D) preserved embryonic tissues, that can also be used for high-resolution topographical analysis of tissue-specific gene-expression patterns.

This article provides a method to isolate pure embryonic tissues from quail and chicken embryos that can be combined to form ex vivo chimeric organs.

배아 조직과 Thymus 예제를 사용 하는 메 추 라 기-닭고기 공상 기관의 형성의 절연

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed ...

In Vitro Culture of Epithelial Cells from Different Anatomical Regions of the Human Amniotic Membrane

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that the amniotic epithelium is a homogeneous layer. The human amnion can be divided into three anatomical regions: reflected, placental, and umbilical. Each region has different physiological roles, such as in pathological conditions. Here, we describe a protocol to dissect human amnion tissue in three sections and maintain it in vitro. In culture, cells derived from the reflected amnion displayed a cuboidal morphology, while cells from both placental and umbilical regions were squamous. Nonetheless, all the cells obtained have an epithelial phenotype, demonstrated by the immunodetection of E-cadherin. Thus, because the placental and reflected regions in situ differ in cellular components and molecular functions, it may be necessary for in vitro studies to consider these differences, because they could have physiological implications for the use of HAEC in biomedical research and the promising application of these cells in regenerative medicine.

This protocol describes the isolation of epithelial cells from different anatomical regions of the human amniotic membrane to determine their heterogeneity and functional properties for possible application in clinical and physiopathological models.

인간 양막의 다른 해부학 적 영역에서 상피 세포의 시험관 내 배양

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that ...

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

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

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

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

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

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

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

조류 배아 판막병 전구 세포의 분리 및 문화

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

This versatile protocol describes the isolation of premigratory neural crest cells (NCCs) through the excision of cranial neural folds from chick embryos. Upon plating and incubation, migratory NCCs emerge from neural fold explants, allowing for assessment of cell morphology and migration in a simplified 2D environment.

병아리 두개골 신경 볏 세포 배양의 준비 및 형태학적 분석

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like ...