이 작품은 PAT에 NIH 교부금에 의해 투자 Ey10540했다.

1. 이리스 세포 배양 <ol><li> 7 newts (PBS의 준비 3 aminobenzoate 메탄 sulfonic의 산성 에틸 0.1 %에 anesthetized)과 칼슘, 마그네슘 무료 행크스 솔루션 (CMF)에서 곳에서 눈알를 수집합니다. </li><li> 장갑을 변경하고 조직 문화 후드에서 작동합니다. </li><li> 3 초 Lugol&#39;s - EtOH의 눈 볼을 소독. </li><li> CMF 2 번 씻으십시오. </li><li> 행크스에 눈을 전송과 절개를 시작합니다. </li><li> 홍채 - 각막 복잡한을 해부하다 및 신경 망막를 제거합니다. </li><li> 행크스의 새로운 그릇에 단지를 전송합니다. </li><li> 지느러미 및 복부 반쪽에 # 15 메스, 별도의 단지를 사용하여. </li><li> 1 ML은 L - 15을 포함하는 접시에 홍채 조각 (최초 복부)와 장소를 제거합니다. </li><li> 15 % (150 UL) dispase의 볼륨 (7.5 단위 / ML 농도)을 추가합니다. </li><li> 27 2 시간 동안 ° C (parafilm와 랩 요리)에 품어. </li><li> 스트 로마 (27시 플레이스 트립신 용액 ° C)에서 IPE 세포를 분리. </li><li> 1.5 ML 튜브에 IPE 세포를 수집합니다. </li><li> 이분에 대한 RT에서 1,000 RPM (실온)에서 원심 분리기, 뜨는 제거합니다. </li><li> 2 분 동안 RT에서 1000 RPM 1 ML 행크스와 원심 분리기와 세척 뜨는 제거합니다. </li><li> 27에서 2 시간 동안 품어 ° C., 1 ML 트립신 솔루션 추가 </li><li> 이분에 대한 RT에서 1,000 rpm으로 원심 분리기는 트립신을 제거하십시오. </li><li> 2 분 동안 1,000 rpm으로 원심 분리기, 씻어 1 ML L - 15을 추가합니다. </li><li> 천천히 위아래로 800 UL L - 15, pipet ~ 10 번 (이상 12 회가 부착을 줄일 수) 추가합니다. </li><li> 플레이트 27 24 잘 콜라겐 코팅 판과 부화에 IPE 세포 ° C 이주 (보통, 4 등의 4 복부 우물이 7 newts에서 얻을 수있다)에 대한. </li><li> 이 단계에서 세포는 유전자 transfection 또는 유도하거나 렌즈 재생을 방해하는 그들의 역할을 결정하는 요인과 치료에 적합합니다. </li></ol> 2. 이리스 세포의 집합 <ol><li> 번호판을 포함 홍채 세포는 부드럽게 400 μl 매체 소용돌이에 dispase 솔루션의 20μl를 추가합니다. </li><li> 27 ° C 야간에 대한 번호판을 품어. </li><li> Pipet 부드럽게 위아래로 세포를 이동시키다하기 </li><li> Eppendorf 튜브 및 RT에서 1,000 rpm으로 2 분 스핀에 플레이스 세포. </li><li> 1 ML 전체 L - 15을 추가하여 중간 및 세척을 제거, RT에서 1,000 rpm으로 2 분 스핀 매체를 제거합니다. </li><li> 집합마다 2000-7000 세포를 사용합니다. 각각의 튜브로 집계 200 UL L - 15을 추가합니다. </li><li> 새로운 eppendorf 튜브에 분할 세포 (200 μl). </li><li> RT 2 분 1,000 rpm으로 회전. </li><li> 27 48 시간을위한 품어 ° C. </li></ol> 3. Aggregrated 세포의 주입 <ol><li> 뉴트의 눈 각막에 슬릿을하고 렌즈를 제거합니다. </li><li> 렌즈 제거 후 눈 복부 측면에 바로 각막 조직 아래 IPE 세포 집계를 놓습니다. </li><li> newts은 그들이 렌즈를 다시 생성하도록 한 달 동안 보관됩니다. </li></ol> 4. 뉴트 아이의 포함 <ol><li> 집계로 이식 뉴트의 눈을 제거하고 인산에 배치 호수 (PBS)가 버퍼. </li><li> 최소 4 시간 또는 밤새 4 ° C에서 4 % paraformaldehyde에 수정. </li><li> 30 분 PBS, 4 ° C에서 씻으십시오. </li><li> 4 ° C, 30 분 0.85 %의 식염수에 그것을 취급합니다. </li><li> 15 분 동안 RT : 살린 / 에탄올 (1:1)에 씻으십시오. </li><li> 70 % 에탄올에 세척 : RT, 15 분. 한번 반복합니다. </li><li> RT 30 분 각 85 % 에탄올과 95 % 에탄올로 세척 </li><li> 100 % 에탄올에 세척 : RT, 30 분. 한번 반복합니다. </li><li> 4 스토어 ° C 또는 계속합니다. </li><li> 100 % 크실렌의 세척 : RT, 30 분. 한번 반복합니다. </li><li> 60 ° C, 45 분 : 크실렌 / 파라핀 (1:1)으로 처리합니다. </li><li> 60 ° C, 20 분 100 % 파라핀과 치료. 그것을 두 번 더 반복합니다. </li><li> 금형을 포함에 삽입할 수 있습니다. </li></ol> 5. Sectioning <ol><li> 마이크로톰를 사용하여 임베디드 눈 15 μm의 섹션을 준비합니다. </li><li> 젤라틴 코팅 슬라이드에 눈 섹션을 장소와 따뜻한 슬라이드에두고. </li></ol> 6. 더럽히는 것 <ol><li> 이곳은 10 분 크실렌에 슬라이드. 한번 더 반복합니다. </li><li> 의 하이드 레이트 : 100 %, 95 %, 80 %, 70 %, 일분 각 30 % 에탄올 </li><li> 잠시 디 물에 헹굼. </li><li> 15 분 동안 PBS로 씻으십시오. </li><li> 15 분 동안 PBST (PBS에 0.​​2 % 트리톤 X - 100)에 씻으십시오. </li><li> 15 분 동안 PBS로 씻으십시오. </li><li> 한시간 솔루션 (10 % 염소 차 항체)를 차단에 놓습니다. </li><li> 하룻밤에 대한 기본 항체에 놓습니다. </li><li> 15 분 동안 PBS의 기본 항체를 씻으십시오. </li><li> 15 분 PBST로 씻으십시오. </li><li> 15 분 동안 PBS로 씻으십시오. </li><li> 어둠 속에서 2 시간 동안 차 항체에 플레이스 (PBS에 희석 1:100). </li><li> 십오분 각 후 마운트를위한 PBS, PBST, 그리고 PBS로 씻으십시오. </li><li> 형광 현미경 슬라이드를 관찰합니다. </li></ol> 7. 대표 결과 : culturing 뉴트 IR의이 절차는는 세포는 지느러미와 복부 IPE 세포의 재생 가능성을 연구하기 위해 활용되었습니다. 또한, 그것은 뉴트 안구의 렌즈 재생 메커니즘으로 기여하는 특정 유전자를 연구하는 것도 가능합니다. 전지은 2 주 동안 교양되었을 경우 (그림 1) 이들은 렌즈에서 재생 기능을 조사하는 유전자로 transfected 수 있습니다. 특히 관심의 복부 아이리스 유발 유전자 수 있습니다. 복부 홍채 세포가 렌즈에 transdifferentiate 수 없기 때문에 (그림 2) 후보 유전자의 유도 기능을 공부하실 수 있습니다. 과거에는이 기법을 사용하는 우리는 여섯 3이 넘는 retinoic 산성 렌즈 유도의 존재로 표현되었을 때 복부 홍채 6 관찰되었다 것으로 나타났습니다. 그림 3에서는 복부 이리스 집계는 지느러미 홍채 (화살표)에서 호스트 렌즈와는 다른이 아닌 완전히 성장과 차별화된 렌즈 (화살촉)에 상승을 준 것을 알 수 있습니다. <img src="/files/ftp_upload/2713/2713fig1.jpg" alt="그림 1" /> 그림 1. A) 도설 홍채 색소 상피 세포는 2 주 동안 체외에서 배양해. B) 복부 홍채 색소 상피 세포는 2 주 동안 체외에서 배양해. 착색은 지느러미와 복부 양쪽 홍채 세포에서이 단계로 계속합니다. <img src="/files/ftp_upload/2713/2713fig2.jpg" alt="그림 2" /> 그림 2. 교양 IPE 세포의 재생 능력. 지느러미 IPE 세포 집계 (화살촉)에서) 렌즈 유도. 지느러미 홍채 (화살표), DI에서 렌즈 유도 호스트 : 지느러미 홍채, 바이 : 복부 홍채, 르 : 렌즈 상피,면 : 렌즈 섬유. 복부 IPE 세포 집계 (화살촉)에서 렌즈 유도의 B) 부재. 지느러미 홍채 (화살표)에서 렌즈를 유도 호스트. <img src="/files/ftp_upload/2713/2713fig3.jpg" alt="그림 3" /> 그림 3. 복부 집계에서 렌즈 유도 여섯 - 3 transfected 및 retinoic 산성과 치료. 지느러미 홍채 (화살표)에서 렌즈를 유도 호스트. 복부 집계 (화살촉)에서 렌즈 유도.

<ol>
	<li>Tsonis, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+in+vertebrates.">Regeneration in vertebrates.</a> Dev Biol. 221, 273-284 (2000).</li><li>Tsonis, P. A., Madhavan, M., Tancous, E. E., Del Rio-Tsonis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+newt's+eye+view+of+lens+regeneration.">A newt's eye view of lens regeneration.</a> Int. J. Dev. Biol. 48, 975-980 (2004).</li><li>Del Rio-Tsonis, K., Tsonis, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eye+regeneration+at+the+molecular+age.">Eye regeneration at the molecular age.</a> Dev. Dyn. , 226-2211 (2003).</li><li>Okamoto, M., Ito, M., Owaribe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Difference+between+dorsal+and+ventral+iris+in+lens+producing+potency+in+normal+lens+regeneration+is+maintained+after+dissociation+and+reaggregation+of+cells+from+the+adult+newt,+Cynops+pyrrhogaster.">Difference between dorsal and ventral iris in lens producing potency in normal lens regeneration is maintained after dissociation and reaggregation of cells from the adult newt, Cynops pyrrhogaster.</a> Develop Growth Differ. 40, 11-18 (1998).</li><li>Hayashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+transfection+system+for+functional+gene+analysis+in+adult+amphibian+lens+regeneration.">Highly efficient transfection system for functional gene analysis in adult amphibian lens regeneration.</a> Develop Growth Differ. 43, 361-370 (2001).</li><li>Grogg, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+inhibition-driven+regulation+of+six-3+underlies+induction+of+newt+lens+regeneration.">BMP inhibition-driven regulation of six-3 underlies induction of newt lens regeneration.</a> Nature. 438, 858-862 (2005).</li><li>Nakamura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNAs+in+Newt+Lens+Regeneration:+Specific+Control+of+Proliferation+and+Evidence+for+miRNA+Networking.">miRNAs in Newt Lens Regeneration: Specific Control of Proliferation and Evidence for miRNA Networking.</a> PLoS One. 5, e12058-e12058 (2010).</li><li>Madhavan, 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+role+of+Pax-6+in+lens+regeneration.">The role of Pax-6 in lens regeneration.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 14848-14853 (2006).</li><li>Maki, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte-type+linker+histone+B4+is+required+for+transdifferentiation+of+somatic+cells+in+vivo.">Oocyte-type linker histone B4 is required for transdifferentiation of somatic cells in vivo.</a> FASEB J. 24, 3462-3467 (2010).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이 프로토콜은 newts에서 렌즈의 재생 메커니즘을 연구하기 위해 체외 시스템을 마련하였습니다. 집계 이후 (중 지느러미 또는 복부가 충실하게 자신이 재생이 기법 중 생체내 행동에 newts에 transgenesis에 필요한과 기능의 이득뿐만 아니라 기능을 실험 7, 8, 9의 손실에 대해 사용할 수있는 엄청난 노력을 경감 할 수 따릅니다. 또한 , 전체 집계 또는 붓꽃은 쉽게 성장 요소로 취급하고이 프로토콜의 설명에 그들의 영향을 조사합니다. 예를 들어 BMP 통로의 역할이 기술 6을 사용 연구되어 수 있습니다.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Lugol's solution</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L-6146</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H-4034</td>
<td>&nbsp;</td>
<tr>
<td>Ethyl 3-aminobenzoate methanesulfonic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E-10521</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>17105-041</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin 1:250</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>27250018</td>
<td>&nbsp;</td>
<tr>
<td>L-15 ( Leibovitz)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L-4386</td>
<td>&nbsp;</td>
<tr>
<td>DNase 1</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D5025-150KU</td>
<td>&nbsp;</td>
<tr>
<td>Kanamycin Sulfate</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>100x, 15160</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F4135</td>
<td>&nbsp;</td>
<tr>
<td>Fungizone (amphotericin B solution)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A2942</td>
<td>&nbsp;</td>
<tr>
<td>24 well collagen coated plate</td>
<td>&nbsp;</td>
<td>BD biosciences</td>
<td>354408</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a system for culturing iris pigment epithelial cells to study lens regeneration in newt

뉴트 및 axolotl 같은 도롱뇽은 팔다리, 척수, 눈, 뇌, 심장, 턱 1 꼬리로의 손실 신체 부위 중 많은 것들을 다시 생성하는 능력을 보유하고 있습니다. 특히, newts는 렌즈 재생 기능에 대해 고유합니다. 렌즈 제거시, 지느러미 홍채의 IPE 세포는 렌즈 세포에 transdifferentiate 결국 한 달 2,3의 새로운 렌즈를 형성하고 있습니다. 중생의이 속성은 복부 홍채 세포에 의해 전시되지 않습니다. 홍채 세포의 재생 가능성은 체외 교양 IPE 세포에의 이식을하여 공부하실 수 있습니다. 문화, 지느러미 및 복부 홍채 세포가 먼저 눈에으로부터 격리되고 이주 (그림 1)의 기간 동안 별도 양식. 이러한 교양 세포 reaggregated와 뉴트 아이로 다시 심어 있습니다. 과거 연구는 복부 집계가 4,5 (그림 2) 따라서 생체내 과정에서 recapitulating, 렌즈를 형성하지 않는 반면, 지느러미 reaggregate는 렌즈를 형성 능력을 유지하는 것으로 나타났습니다. 지느러미 및 복부 홍채 세포의 재생 가능성을 결정하는이 시스템은 렌즈 재생에 관련된 유전자 및 단백질의 역할을 공부에 매우 유용합니다.

Watch this Scientific Journal Video about A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt at JoVE.com

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

뉴트에서 렌즈는 홍채 색소 상피 세포 (IPEs)의 transdifferentiation하여 지느러미 홍채에서 항상 다시 생성합니다. 여기서 우리는 뉴트의 눈에 문화 지느러미와 복부 뉴트 IPE 세포와 그 이식하는 절차를 설명합니다. 이식 세포 그런 다음 조직 sectioning과 immunohistochemistry으로 공부하고 있습니다.

뉴트의 렌즈 재생을 공부 Culturing 이리스 안료 상피 세포에 대한 시스템

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

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Research

JoVE Journal

Biology

11.7K 조회수.  University of Dayton. 뉴트에서 렌즈는 홍채 색소 상피 세포 (IPEs)의 transdifferentiation하여 지느러미 홍채에서 항상 다시 생성합니다. 여기서 우리는 뉴트의 눈에 문화 지느러미와 복부 뉴트 IPE 세포와 그 이식하는 절차를 설명합니다. 이식 세포 그런 다음 조직 sectioning과 immunohistochemistry으로 공부하고 있습니다.

비디오: A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

Zebrafish의 Pronephros의 레이저 절제는 신장 상피 조직이 재생된를 공부

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

연구

생물학

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

활액 유체에 Culturing 인간 관절 Chondrocytes을위한 3D 시스템

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Cryopreserved 샘플 복구를 위해 손수 함 장치 실수로 극저온 저장 탱크 떨어졌고

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

헤어 재생시 상피 - 중간 엽 시그널링의 빠른 유전 분석

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

간엽 전환을 유도하고 상피의 분석

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

에어 리퀴드 인터페이스에서 인간의 비강 상피 세포의 배양

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

사용<em&gt; 예쁜 꼬마 선충</em&gt; 다세포 생물의 단백질 항상성을 연구하는 모델 시스템으로

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Zebrafish의에서 콩팥 세포 독소 미세 주입은 급성 신장 손상을 모델링하기

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Intestinal Epithelial Regeneration in Response to Ionizing Irradiation

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the active proliferation of intestinal stem cells located at the bottom of intestinal crypts. However, during events of acute intestinal injury, these active intestinal stem cells undergo cell death. Gamma irradiation is a widely used colorectal cancer treatment, which, while therapeutically efficacious, has the side effect of depleting the active stem cell pool. Indeed, patients frequently experience gastrointestinal radiation syndrome while undergoing radiotherapy, in part due to active stem cell depletion. The loss of active intestinal stem cells in intestinal crypts activates a pool of typically quiescent reserve intestinal stem cells and induces dedifferentiation of secretory and enterocyte precursor cells. If not for these cells, the intestinal epithelium would lack the ability to recover from radiotherapy and other such major tissue insults. New advances in lineage-tracing technologies allow tracking of the activation, differentiation, and migration of cells during regeneration and have been successfully employed for studying this in the gut. This study aims to depict a method for the analysis of cells within the mouse intestinal epithelium following radiation injury.

The gastrointestinal tract is one of the most sensitive organs to injury upon radiotherapeutic cancer treatments. It is simultaneously an organ system with one of the highest regenerative capacities following such insults. The presented protocol describes an efficient method to study the regenerative capacity of the intestinal epithelium.

이온화 조사에 대한 반응으로 장 상피 재생

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

돼지 망막 색소 상피 세포의 1 차 배양

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...