우리는 활액 유체 저장 및 원심 분리와 도움을 제공하는 로빈 Nye (Tufts 의료 센터), Tomoya Uchimura와 다나 케언즈 (Tufts 대학)을 감사드립니다. 이 작품은 출발지에 대한 NIH (1R01AR059106 - 01A1)에 의해 추진하는 사업

활액 액체에 culturing 인간의 관절 chondrocytes을위한 3D 시스템 이 작품에서 우리는 수정된 제조 - 제안 캡슐화 프로토콜 (Lonza, 31)를 사용 알지네이트요 비즈 인간의 관절 chondrocytes를 캡슐. 이러한 3 차원 구조를 사용하여, 우리는 인간 활액 액체의 다양한 비율을 포함하는 문화 매체 culturing 전지 시스템을 개발 및 연골의 유전자 발현에 대해 이러한 3D 구조를 평가했습니다. 1. 입체 (3D) 캡슐에 대한 인간의 관절 chondrocytes (HAC)를 준비 <ol><li> 37 일 분 ° C의 물을 욕조에 인간의 관절 chondrocytes (HAC) (Lonza) (항로 2) 유리병 (1 ML)을 녹여. </li><li> chondrocyte 성장 미디어 (Lonza)와 세포를 수집하는 3-5 분 3,000 rpm으로 원심 분리기의 1ml와 함께 섞는다. 뜨는 폐기하십시오. </li><li> chondrocyte 성장 미디어 Resuspend 세포 펠렛 (Lonza). </li><li> 10cm 조직의 플레이트 세포문화 플레이트 (BD Biosciences) 및 chondrocyte 성장 미디어 (Lonza)의 문화가 세포가 합류 때까지. HACs없이 높은 세 이상 또는 4 (P3 또는 P4) 통로 번호를 사용해야합니다. </li><li> 알지네이트요 비드 캡슐에 대한 세포를 수집하는 trypsinzation 다음 155mM NaCl 대신 표준 PBS와 함께 접시에 HACs 씻으십시오. 일단 세포가 분리되며, 5 분에 대한 3000rpm에서 세포를 다운 스핀. 세포는 이제 3D 캡슐에 대한 준비가되어 있습니다. </li></ol> 2. 3D 구슬로 HACs를 캡슐 8x10 5 셀 / ML의 밀도에 1.2 % 알지네이트요 솔루션 (시그마)의 HACs을 Resuspend. 휴대폰 번호는 표준 셀 카운터를 사용하기 전에 캡슐로 결정됩니다. 그것은 구슬에있는 세포의 균등을 보장하기 위해 잘 혼합하는 것은 매우 중요합니다. <ol><li> 피펫 22 게이지 바늘 (Tyco 의료, 주식 회사)에 연결된 12 ML 주사기로 HAC / 알긴산 솔루션 혼합물. </li><li> 한편, 102m와 50 ML의 비커를 준비볼륨에서 M CaCl 2 5 회 HAC / aginate 솔루션의. 비커 안에 저어 막대를 놓고 (약 150 RPM에서) 천천히 102 MM CaCl 2 용액을 저어. </li><li> CaCl 2 용액의 표면 위에 육인치에 대한 주사기의 끝부분을 잡고 CaCl 2 솔루션 dropwise로 HAC / 알긴산 혼합물을 추가합니다. 일반적으로 결과 알지네이트요 비즈는 약 10 4 세포 / 비드의 캡슐 밀도와 직경 2mm입니다. 참고 : CaCl 2 용액을 통해 주사기의 높이는 중요합니다. 짧은 거리는 고르지 않은 기계적 무결성과 세포를 캡슐 화 원인, 원형 모양의 구슬 대신 모양의 눈물 드롭집니다. </li><li> 비즈가 20-25 분 CaCl 2 용액에서 저어 보자. 다른 알지네이트요의 캡슐화 프로토콜은 10 분 정도의 짧은 배양 시간을 나타내는 반면, 우리는 CaCl 2 용액과 함께 더 이상 부화 시간이 될의 무결성을 향상 발견광고. </li><li> CaCl 2 용액을 제거 chondrocyte 차별화 매체 (Lonza)에 한 다음 2-3 번 NaCl로부터 2-3 권에 비즈를 씻어합니다. 다른 알지네이트요 비드 캡슐 프로토콜은 위의 세탁 절차 필터를 사용하여 포함하는 동안, 우리는 알지네이트요 비즈가 종종 따라서 캡슐의 효율성을 감소, 필터 및 드라이 아웃에 갇혀되고있는 것으로 나타났습니다. 우리는 가장 효율적인 비즈가 폐기되기 전에 비커의 바닥에 대한 해결책을 해결하도록 발견했습니다. </li><li> 문화 요리에 비즈를 전송하는 표준 주걱을 사용합니다. 우리는 일반적으로 문화를 잘 chondrocyte 성장 매체 2 일간 3cm 당 12 구슬을 chondrocytes들이 전에 활액 액체와 chondrocyte 차별화 매체 (Lonza)에 스위칭 성장 있던 매체 캡슐 프로세스 조정할 수 있습니다. </li></ol> 3. 활액 유체 문화 매체 문화 chondrocytes <ol><li> 신선한 활액 액체는 O에서 구할 수 있습니다utpatient 클리닉 (우리는 Tufts 의료 센터에서 활액 액체를 취득). 활액 액체는 실험실로 15ml 팔콘 튜브에 전송하고, 즉시 세포 파편을 제거하는 15을 위해 3000 RPM에서 centrifuged 수 있습니다. 1.5 ML의 microcentrifuge 튜브로 나누어지는는 세포 무료 활액 액체, 동결 - 해동 반복되지 않도록합니다. 뜨는는 -80 ° C에서 사용까지 저장할 수 있습니다. </li><li> 이전 culturing하기 위해 지속적인 100mM 아스코르비 산의 농도와 9 밀리 CaCl 2 (CaCl 2의이 추적 금액 알지네이트요의 무결성을 보장하는 것입니다과 체적 비율을 변화시 chondrocyte 차별화 매체와 활액 유체 (CDM, Lonza)를 혼합 구슬). </li><li> 37 ° C 배양기에서 락을 플랫폼에서 번호판을 유지 (출렁 이는 주파수 : 약 75 회 / 분) 알지네이트요 비즈 내에서 영양 배포를 도와, 그리고 구슬의 clumping 줄일 수 있습니다. 이것은 장시간 (최대 4 주)에 대한 우리 문화의 이러한 chondrocytes와 같이 특히 중요합니다. 주의: chondrocyte 성장과 차별화 미디어 (Lonza는) 2 일반적으로 사용되는 항생제 gentamycin과 amphotericin를 포함하는 동안 우리는 chondrocyte culturing에 대한 활액 액체의 다른 비율을 사용할 때, 우리는 어떤 항생제를 보충하지 않았다. 오염이도 관찰되지 않았습니다. </li><li> 모든 2-3일 미디어 혼합물을 변경합니다. 활액 유체의 점도는 장기적인 문화 중 알지네이트요 비즈에서 캡슐 culturing의 chondrocytes의 주요 장애물이었다. 우리는 또한 알지네이트요 비즈의 무결성을 강화 102mM CaCl 2 (50 % V / V)와 활액 유체 - 보충 미디어를 희석하는 것이 가장 효과적인 발견했습니다. 이러한 방법으로, 매체는 천천히 구슬을 손상시키지 않고, pipet로 제거할 수 있습니다. </li></ol> 4. 유전자 발현 분석을위한 알지네이트요 비즈에서 수확 HACs 유전자 발현 분석을위한 알지네이트요 비즈에서 HACs을 수확 때 특별한주의가 있어야합니다. <ol><li> 알지네이트요 비즈 - 3 TI 씻으십시오약 5 분 때마다 102 MM CaCl 2 대. </li><li> 4-5 번 그 구슬의 볼륨에서 55 밀리미터의 NaCitrate을 추가하여 chondrocytes를 검색하십시오. 알지네이트요 비즈가 완전히 NaCitrate 솔루션에 열중하는 것이 중요합니다. 흔들거나 20-30 분 바위. </li><li> 세포를 돌려서 뜨는 폐기하십시오. </li><li> RT - PCR 분석을 위해, 세포 세포 용해 버퍼의 펠릿 (Qiagen RNA 격리 키트) 및 RNA 정화 진행을 resuspend. </li></ol> 5. histological 분석을 위해 알지네이트요 비즈에서 HACs을 수정 특별한주의가 histological 분석을위한 3D 비즈에서 HACs을 수확하기 위해주의가 필요하다. <ol><li> 5 분 약 때마다 102 MM CaCl 2의 알지네이트요 비즈에게 3 번 씻으십시오. 완전히 점성 유체 활액을 멀리 세척하기 위해, 우리는 37 ° C 배양기 (출렁 이는 주파수에 락을, 야간 chondrocyte 차별화 매체 (Lonza)로 세척하는 것이 가장 효과적인 발견 : 약 75 시간S / 분). chondrocyte 성장 매체는이 연장 씻어에서 chondrocyte 생존을 보장하고 고정 에이전트의 최대 침투를 허용하는 데 사용되었다. </li><li> 야간 70 % 에탄올에 비즈를 수정하고, histological 분석 진행합니다. Dapi의 얼룩을 수행하려면, 부드러운 락을 미만 1 시간에 대한 Dapi 솔루션 (500ng/ml)와 알지네이트요 비즈를 품어, 15 분 102mM CaCl 2 때마다 3 번 씻는다. Dapi 이미지는 다음 형광 현미경으로 볼 수 있습니다. 구슬은 alcian 파란색과 H &amp; E의 얼룩에 대한 sectioned 수 있습니다. </li></ol> 6. 대표 결과 활액 액체의 높은 비율 인간의 chondrocytes위한 3D culturing 방법은 Fig.1에 표시된 설계도 다이어그램에 그려져 있습니다. 인간 chondrocytes가 알지네이트요 비즈에서 캡슐 후에, 그들은 활액 액체의 비율을 변화와 보완 매체 성장할 수있었습니다. 때문에 활액 유체의 점도, 그것은하는 것이 필수적입니다문화 chondrocytes는 지속적인 락을 조건 아래에있는 연골 구조의 clumping을 방지하고 영양도 배포를 보장합니다. 이것은 고정 또는 용해 버퍼 (Fig.1) 비즈 침투 수 있도록 chondrocytes을 검색하기 전에 광범위하게 알지네이트요 비즈를 씻어도 필수적입니다. 알지네이트요 비즈의 chondrocytes의 명시야 (BF) 이미지 Fig.2에 표시됩니다. Dapi의 얼룩은 비즈 내에서 세포의 usniform 분포 (그림 2) 확인 수행되었다. 21 일 문화 기간의 끝에, 유전자 표현 분석 qRT - PCR에 의해 수행되었다. 그것이 chondrocytes 32 qPCR 분석을위한 가장 안정적인 레퍼런스 유전자 중 하나가 될하기로 결정했습니다로서 참조 유전자 GAPDH는 모든 PCRs에 대한 정상화를 위해 사용되었다. 예제는 그림에 표시됩니다. 우리가 관절염 여섯 환자에서 풀링된 활액 액체의 교양 인간의 관절 chondrocytes에서 결과를 분석 3. 사실과 일치와는 chondrocytesLonza는 불가피하게 취소 분화 33 이어질 것이 2D 문화에서 확장되었습니다, 우리는 그런 일 0 chondrocytes는 연골 매트릭스 유전자의 최소한의 수준 (그림 3) 표현 발견했습니다. 활액 액체로 보충 chondrocyte 차별화 매체 (Lonza)이나 매체 chondrocytes의 3D culturing 크게 날 21 일 (Fig.3) 34에 의해 다시 차별 chondrocyte을 나타냅니다 콜라겐, aggrecan 및 MMP13의 연골의 유전자 발현을 증가시켰습니다. 미디어의 활액 액체의 비율을 증가 시키면 연골 매트릭스 마커 콜라겐 II ​​및 aggrecan의 mRNA 발현 (Fig.3A 및 3B)의 비교 수준의 결과. 중간 혼자 (Fig.3C)에서 교양 이들과 비교뿐만 아니라, 100 % 활액 액체의 양식 chondrocytes도 연골 저하 효소 MMP13 mRNA 표현의 감소를 전시. 흥미롭게도, 세포 죽음이 표시 caspase 3의 표현 수준은 점차 제안, 활액 액체의 비율 증가와 함께 감소되는의ynovial 유체 culturing 감소 apoptosis 수준 (그림 3D)으로되었다. 따라서, 우리의 결과는 3D 환경에서 활액 액체 높은 수준의 culturing 인간의 관절 chondrocytes이 가능한 기술입니다 보여줍니다. 테이블과 피규어 <img alt="그림 1." src="/files/ftp_upload/3587/3587fig1.jpg" /> 그림 1. 3D 알지네이트요 비즈의 활액 액체의 높은 비율의 문화 인간의 관절 chondrocytes에 대한 방법의 도식 다이어그램. 첫째, chondrocytes 및 알지네이트요 솔루션은 혼합 수 있습니다. CaCl 2 솔루션 드롭 현명 적용하면, chondrocytes는 가교 CA - 알지네이트요의 히드로겔 비즈 내에서 움직일 수 있습니다. 이러한 3D 구조는 다음 인간 활액 액체의 비율을 변화와 chondrocyte 차별화 매체 (Lonza)에 양식이 있습니다. 락을 조건 culturing 21 일 후에 세포를 포함하는 알지네이트요 비즈는 세포가 유전자에 대한 검색되는 이후에 광범위하게 씻어 아르표현 분석. <img alt="그림 2." src="/files/ftp_upload/3587/3587fig2.jpg" /> 그림 2. 브라이트 필드 (BF)와 인간의 관절 chondrocytes의 Dapi 이미지 0퍼센트, 30 %, 50 %, 70 % 및 100 % 활액 액체와 캡슐 문화. Chondrocytes 모든 문화 조건에서 모양 구형했다. 명시야 (BF)와 Dapi의 이미지는 chondrocytes의 위치와 분포를 확인하기 위해 입혔다했다. Insets, 확대 이미지를 표시합니다. 화살촉, BF와 Dapi의 얼룩 이미지의 chondrocytes의 colocalization. <img alt="그림 3." src="/files/ftp_upload/3587/3587fig3.jpg" /> 그림 3. 하루 캡슐 인간의 관절 chondrocytes의 qRT - PCR 분석 0 (D0) 활액 액체 (SF) (0 %, 30 %, 50 %, 70 다양한 비율로 보충 매체 culturing (D21) 21 일 후에 % 및 100 %). 네 독립 표본의 결과는 여기에 표시됩니다. GAPDH는 모든 PCRs에 대한 내부 참조로 사용되었다. <str긴 사연이&gt;. 콜라겐 II의 mRNA의 표현. B. Aggrecan의 mRNA의 표현. C. MMP13 mRNA의 표현. D. Caspase 3 mRNA의 표현. 통계 중요성은 일 0 샘플과 0 %의 주 21 샘플 및 INSTAT 소프트웨어를 사용 백퍼센트 활액 액체 (SF) culturing를 평가했다. * 나타냅니다 P &lt;0.05.

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우리는 공개 아무것도 없어.

이 보고서에서는, 우리는 인간 활액 액체의 높은 농도를 포함 매체 3D 환경에서 인간의 관절 chondrocytes의 문화에 대해 수있는 방법을 개발했습니다. 활액 액체는 관절 chondrocytes이있는 공동 구멍에 자연 환경을 구성하는 주요 구성 요소 중 하나입니다. 그러나, 활액 유체의 점도는 chondrocytes의 세 차원 장기 culturing에 대한 큰 도전을했습니다. 3D 막아야 환경에서 구축하고 방지하기 위해 집계에서도 ?...

특정 시약 및 장비의 테이블 : <table border="1"><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> 알긴산 (Alginic 산성 나트륨 소금) </td><td> 시그마 </td><td> A2158 - 250G </td><td> 2.4 % 용액은 40 ° C에 보관 </td></tr><tr><td> 염화칼슘 이수화물, 세분화된 </td><td> JT 베이커 </td><td> A19339 </td><td></td></tr><tr><td width="239" rowspan="2" > Chondrogenic 성장 미디어 </td><td width="254" rowspan="2" > Lonza </td><td width="182" > CC - 3156 (기본 미디어) </td><td width="141" rowspan="2" ></td></tr><tr><td width="182" > CC - 4409 (보충) </td></tr><tr><td width="239" rowspan="2" > Chondrogenic 차별화 미디어 </td><td width="254" rowspan="2" > Lonza </td><td width="182" > CC - 3226 (기본 미디어) </td><td width="141" rowspan="2" ></td></tr><tr><td width="182" > CC - 4408 (보충) </td></tr><tr><td> 인간 관절 chondrocytes </td><td> Lonza </td><td > CC - 2550 </td><td></td></tr><tr><td> Dapi (4 &#39;, 6 Diamidino - 2 - phenylindole의 dihydrochloride) </td><td> 시그마 - 올드 리치 </td><td > D9542 </td><td></td></tr><tr><td> RNeasy 미니 키트 (RNA 추출을위한) </td><td> Qiagen </td><td > 74,104 </td><td></td></tr><tr><td> PCR 시약 : SYBR 녹색 </td><td> 콴타 </td><td > 95053-500 </td><td></td></tr><tr><td> 12 ML의 주사기 </td><td> Tyco - 켄달 - Monoject </td><td > 512,852 </td><td></td></tr><tr><td> 22 - Gague의 피하 니들 </td><td> Tyco - 켄더나 - Monoject </td><td > 8881 </td><td></td></tr><tr><td> 현미경 </td><td> 올림포스 산 </td><td > IX71 </td><td></td></tr><tr><td> 플랫폼 로커 </td><td> Thermoscientific thermolyne </td><td > Vari - 믹스 </td><td></td></tr><tr><td></td><td></td><td ></td><td></td></tr><tr><td> Primers 시퀀스 </td><td></td><td ></td><td></td></tr><tr><td> 콜라겐 IIa - 앞으로 </td><td> 5&#39; - TTC ATC CCA CCC TCT CAC AGT - 3 &#39; </td><td ></td><td></td></tr><tr><td> 콜라겐 IIa - 역방향 </td><td> 5&#39; - CCTCTGCCTTGACCCGAA - 3 &#39; </td><td ></td><td></td></tr><tr><td> MMP13 - 앞으로 </td><td> 5&#39; - TGT GCC CTT CTT CAC ACA GAC ACT - 3 &#39; </td><td ></td><td></td></tr><tr><td> MMP13 - 역방향 </td><td> 5&#39; - AGA AGC 개그 CTT TGA GTC AT &amp; T GCC - 3 &#39; </td><td ></td><td></td></tr><tr><td> Caspase 3 - 앞으로 </td><td> 5&#39; - TCA TTA TTC AGG CCT GCC GTG GTA - 3 &#39; </td><td ></td><td></td></tr><tr><td> Caspase 3 역 </td><td> 5&#39; - TGG ATG AAC 편대의 편대 장의 개그 CCA TCC TTT의 -3 &#39; </td><td ></td><td></td></tr></table>

a 3d system for culturing human articular chondrocytes in synovial fluid

연골 파괴는 관절염의 중심 병적인 기능, 미국에서 장애의 주요 원인이다. 성인의 연골은 생체내 매우 효과적으로 재생되지 않으며, 그 결과로 관절염은 연골 손실을 돌이킬에 이르게하고 만성 통증과 부동의 1,2와 함께합니다. 연골 조직 공학 조직 기능을 재생 및 복원하기 위해 유망한 가능성을 제공합니다. 이 기술은 일반적으로 결과 3D 엔지니어링의 목표 생체내 3-6 결함 사이트에 이식 수 화학적 및 biomechanically 성숙한 조직과 일정 기간 동안 균형 매체 만드는 천연 또는 합성 공사장 공중 발판과 culturing에 시딩 chondrocytes을 포함 . chondrocyte 성장과 매트릭스 증착을위한 최적의 조건을 달성하면 연골 조직 공학의 성공을 위해 필수적입니다. 북극의 원시 공동 캐비티, 연골에뼈 ular 표면은 활액 액체에서 목욕을합니다. 이 명확하고 점성 유체의 avascular 관절 연골에 영양분을 제공하고 성장 요인, 크린 시토킨 및 chondrocyte 신진 대사를 7,8에 대한 중요한 효소가 포함되어 있습니다. 또한, 활액 액체는 주로 secreting 두 가지 핵심 구성 요소, hyaluronan과 lubricin 9 10를 통해 연골 표면 사이의 낮은 마찰 운동을 용이하게합니다. 반면, 조직 공학 연골은 가장 자주 인공 미디어 양식입니다. 이러한 미디어 가능성이 chondrocyte 신진 대사를 공부에 대한 더 정의된 조건을 제공할 수 있지만, 활액 액체는 대부분 정확하게 관절 chondrocytes 안으로 거주하는 자연 환경을 반영 사실, 활액 액체가 얻고 저장하기 위해 쉽게되는 장점을 가지고 있으며, 자주 정기적으로 기관 보충함 수 있습니다. 몇몇 그룹은 성장 인간, 소, 토끼 및 개 C에서 활액 액체와 문화 매체를 보충해야hondrocytes하지만, 대부분의 활액 액체 (아래 20 %) 11-25 만 낮은 수준을 사용합니다. 닭, 말과 인간 chondrocytes는 활액 액체의 높은 비율과 매체 교양되었습니다 있지만, 이러한 문화 시스템은 2 차원 26-28되었습니다. 여기는 21 일 기간 동안 활액 액체의 높은 비율 (최대 100 %)과 3D 시스템에 culturing 인간의 관절 chondrocytes 우리의 방법을 제시한다. 이렇게, 우리는 활액 액체의 높은 점도로 표시 주요 장애물을 극복. 이 시스템은 더 이상을위한 자연 환경을 모방하는 연골에 대한 자연 환경을 구성하는 두 개의 다른 중요한 요소 (산소 긴장과 기계적 부하) 29,30와 함께 할 수있는 3D 설정에서 활액 액체 인간의 chondrocytes 공부의 가능성을 제공합니다 연골 성장. 또한,이 시스템은 또한 활액 chondrocytes에서 유체의 활동을 시금 사용 및 개발을위한 플랫폼을 제공 수 있습니다연골 재생 기술과 관절염에 대한 치료 옵션을 제공합니다.

Watch this Scientific Journal Video about A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid at JoVE.com

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

활액 액체 높은 수준의 culturing 인간의 관절 chondrocytes의 3D 시스템이 설명되어 있습니다. 활액 액체는 관절 연골의 가장 자연 microenvironment을 반영하고, 쉽게 얻은하고 저장할 수 있습니다. 이 시스템은 따라서 연골 재생을 공부 및 관절염 치료를위한 심사 치료제 사용할 수 있습니다.

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 ...

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20.7K 조회수.  Tufts University School of Medicine. 활액 액체 높은 수준의 culturing 인간의 관절 chondrocytes의 3D 시스템이 설명되어 있습니다. 활액 액체는 관절 연골의 가장 자연 microenvironment을 반영하고, 쉽게 얻은하고 저장할 수 있습니다. 이 시스템은 따라서 연골 재생을 공부 및 관절염 치료를위한 심사 치료제 사용할 수 있습니다.

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

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

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, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

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, ...

연구

생물학

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

작은 분자와 유도 Pluripotent 인간 배아 줄기 세포로부터 인간 심장 엽 성의 전구 물질과 Cardiomyocytes의 효율 유도

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

위한 시스템<em&gt; 예 생체</em배아 췌장의&gt; 배양

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

Internal organs such as the heart, brain, and gut develop left-right (LR) asymmetries that are critical for their normal functions1. Motile cilia are involved in establishing LR asymmetry in vertebrate embryos, including mouse, frog, and zebrafish2-6. These 'LR cilia' generate asymmetric fluid flow that is necessary to trigger a conserved asymmetric Nodal (TGF-&beta; superfamily) signaling cascade in the left lateral plate mesoderm, which is thought to provide LR patterning information for developing organs7. Thus, to understand mechanisms underlying LR patterning, it is essential to identify genes that regulate the organization of LR ciliated cells, the motility and length of LR cilia and their ability to generate robust asymmetric flow.
In the zebrafish embryo, LR cilia are located in Kupffer's vesicle (KV)2,4,5. KV is comprised of a single layer of monociliated epithelial cells that enclose a fluid-filled lumen. Fate mapping has shown that KV is derived from a group of ~20-30 cells known as dorsal forerunner cells (DFCs) that migrate at the dorsal blastoderm margin during epiboly stages8,9. During early somite stages, DFCs cluster and differentiate into ciliated epithelial cells to form KV in the tailbud of the embryo10,11. The ability to identify and track DFCs&mdash;in combination with optical transparency and rapid development of the zebrafish embryo&mdash;make zebrafish KV an excellent model system to study LR ciliated cells.
Interestingly, progenitors of the DFC/KV cell lineage retain cytoplasmic bridges between the yolk cell up to 4 hr post-fertilization (hpf), whereas cytoplasmic bridges between the yolk cell and other embryonic cells close after 2 hpf8. Taking advantage of these cytoplasmic bridges, we developed a stage-specific injection strategy to deliver morpholino oligonucleotides (MO) exclusively to DFCs and knockdown the function of a targeted gene in these cells12. This technique creates chimeric embryos in which gene function is knocked down in the DFC/KV lineage developing in the context of a wild-type embryo. To analyze asymmetric fluid flow in KV, we inject fluorescent microbeads into the KV lumen and record bead movement using videomicroscopy2. Fluid flow is easily visualized and can be quantified by tracking bead displacement over time.
Here, using the stage-specific DFC-targeted gene knockdown technique and injection of fluorescent microbeads into KV to visualize flow, we present a protocol that provides an effective approach to characterize the role of a particular gene during KV development and function.

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer&#039;s Vesicle

Cilia-generated fluid flow in Kupffer&#x2019;s Vesicle (KV) controls left-right patterning of the zebrafish embryo. Here, we describe a technique to modulate gene function specifically in KV cells. In addition, we show how to deliver fluorescent beads into KV to visualize fluid flow.

Kupffer의 소포의 유전자 기능 및 속눈썹 생성 된 유체 흐름의 시각화 분석

Internal organs such as the  heart, brain, and gut develop left-right (LR) asymmetries that are critical for  their normal functions1. Motile cilia are ...

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 ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

갭 접합부 세포 간 통신 : 천연 제품의 독성 물질과 독소의 반작용을 평가하기위한 기능성 바이오 마커, 보건 혜택

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

총회와 &#39;전단 반지&#39;의 응용 프로그램 : 궤도, 단방향 및 정기 유체 흐름 및 전단 응력에 대한 소설 내피 모델

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

마그네틱 활성화 전략을 분리 하 고 정화 활 액 액체 파생 된 중간 엽 줄기 세포는 토끼 모델에서 정렬 셀

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is compartmentalized into crypts and villi: the stem and proliferating cells are confined to the crypts, whereas the villi epithelium contains only differentiated cells. Hence, the normal intestinal crypts, but not the villi, can give rise to organoids in 3D cultures. The procedure described here is applicable only to villus epithelium undergoing dedifferentiation leading to stemness. The method described uses the Smad4-loss-of-function:&#946;-catenin gain-of-function (Smad4KO:&#946;-cateninGOF) conditional mutant mouse. The mutation causes the intestinal villi to dedifferentiate and generate stem cells in the villi. Intestinal villi undergoing dedifferentiation are scraped off the intestine using glass slides, placed in a 70 &#181;m strainer and washed several times to filter out any loose cells or crypts prior to plating in BME-R1 matrix to determine their organoid-forming potential. Two main criteria were used to ensure that the resulting organoids were developed from the dedifferentiating villus compartment and not from the crypts: 1) microscopically evaluating&#160;the isolated villi to ensure absence of any tethered crypts, both before and after plating in the 3D matrix, and 2) monitoring the time course of organoid development from the villi. Organoid initiation from the villi occurs only two to five days after plating and appears irregularly shaped, whereas the crypt-derived organoids from the same intestinal epithelium are apparent within sixteen hours of plating and appear spherical. The limitation of the method, however, is that the number of organoids formed, and the time required for organoid initiation from the villi vary depending on the degree of dedifferentiation. Hence, depending upon the specificity of the mutation or the insult causing the dedifferentiation, the optimal stage at which villi can be harvested to assay their organoid forming potential, must be determined empirically.

The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

장내 빌리 상피에서 오르가노이드의 3D 배양

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...