우리는 설치 및 mStrawberry OP9 세포 라인의 테스트 U. Ganapati 감사합니다. 또한, 우리는 공동 문화와 H. Shafifor 한테 도움을 주셔서 감사합니다. HP는 국립 심장, 폐, 및 혈액 연구소 (F31HL087714) (HP)의 화목에 의해 지원됩니다. 이 작품의 내용은 전적으로 저자의 책임이며 반드시 국립 심장, 폐, 그리고 혈액 연구소 또는 국립 보건원 (NIH)의 공식 견해를 대변하지 않습니다. UCLA의 유동세포계측법 핵심 시설은 NIH (CA - 16042 및 AI - 28697), 존슨 암 센터, UCLA 에이즈 연구소와 의학의 UCLA 학교에서 지원됩니다.

1. ES 세포의 준비 <ol><li> 특정 세포 라인에 대한 표준 프로토콜에 따라 문화 ES 세포. 일 0 mStrawberry OP9 세포의 접시에 충분한 ES 세포를 준비하십시오 (ES 세포는 일 0 cm 2 당 1x10 ^ 3 세포에서 도금됩니다) </li></ol> 2. OP9 세포의 준비 <ol><li> 성장 mStrawberry OP9 세포 전에 OP9 성장 미디어를 준비 OP - 9 세포 배양 매체 <table border="1"><tbody><tr><td></td><td> 재고 </td><td> 최종 CONC. </td><td> 볼륨 (ML) </td></tr><tr><td> aMEM </td><td></td><td></td><td> 39.5 </td></tr><tr><td> FBS (OP - 9 테스트) </td><td> 백퍼센트 </td><td> 20% </td><td> 10 </td></tr><tr><td> L - 글루타민 </td><td> 100X (200mM) </td><td> 2mM </td><td> 0.5 </td></tr><tr><td></td><td></td><td></td><td> 50mL </td></tr></tbody></table> 4 스토어 ° C와 준비 후 일주일 이내에 사용 </li><li> mStrawberry OP9 세포를 유지하기 위해, 미디어, 2 일마다 변경해야하며 세포는 사전 (confluency 70~90%에서 subculture) confluency 도달 세포 subcultured해야합니다. 이 adipocytes에 자신의 차별을 초래할 수 있으므로 mStrawberry OP9 세포 문화는 confluency 90% 벗어났소되지 않습니다. 그림 3을 참조하십시오. <ol class="lalpha"><li> PBS (또는 트립신 - EDTA (에틸렌 다이아 민 테트라 초산)과 PBS + 0.1 % 1X와 1X)와 배 세척 세포 Subculture 세포 </li><li> 37 미리 예열 0.1 % 트립신 - EDTA (에틸렌 다이아 민 테트라 초산)을 추가 ° 5 분 C (또는 셀 분리까지) </li><li> 세포가 분리되면 OP - 9 성장 매체를 추가합니다. 50mL 원뿔 튜브에 세포를 전송합니다. </li><li> 상온에서 5 분 300 XG에 펠렛 전지는. 뜨는 폐기하십시오. </li><li> 신선한 mStrawberry OP9 성장 매체 Resuspend 세포 </li><li> 10 4 세포 / cm 2의 최소 밀도에서 플레이트 세포 </li><li> 공동 culturing의 ES 세포 (주 0, 5 일, 또는 실험의 Day8 / 9)에 필요한 일 이전에 삼일 시작 confluency에 mStrawberry OP9 세포 성장 </li></ol></li></ol> 3. ES / mStrawberry OP9 공동 문화 <ol><li> 공동 문화 차별 미디어 준비 <table border="1"><tbody><tr><td></td><td> 재고 </td><td> 최종 </td><td> 볼륨 (ML) </td></tr><tr><td> aMEM </td><td></td><td></td><td> 39.5 </td></tr><tr><td> FBS </td><td> 백퍼센트 </td><td> 20% </td><td> 10 </td></tr><tr><td> L - 글루타민 </td><td> 200mM </td><td> 2mM </td><td> 0.5 </td></tr><tr><td></td><td></td><td></td><td> 50mL </td></tr></tbody></table></li><li> mStrawberry OP9 세포 (데이 -3) <ol class="lalpha"><li> 항상 주 -3에 OP9 세포가 공동 문화의 주 0 mStrawberry OP9 세포의 합류 레이어를 가지고 있도록 Subculture. 그림 4B를 참조하십시오. 일 0 confluency에 대한 1x10 4 / cm 2 판 mStrawberry는 OP9 세포. 5 일 및 공동 문화의 지속을위한 일 9분의 8에 대한 추가 subculture 세포를해야합니다. </li></ol></li><li class="jove_content"> ES / mStrawberry OP9 공동 문화 설정 (데이 0) <ol class="lalpha"><li class="jove_content"> PBS로 2X ES 세포 와시 </li><li> 5 분 @ 37에 대한 사전 예열 0.25 % 트립신 - EDTA (에틸렌 다이아 민 테트라 초산), 부화와 ES 세포를 Trypsinize ° C </li><li> 세포에 공동 문화 차별 미디어 추가 </li><li> 400xg에서 펠렛 ES 세포, 5 - 7min, RT. 뜨는 취소 </li><li> 공동 문화 차별화 미디어에 Resuspend 세포 </li><li> mStrawberry OP9 세포에서 미디어를 제거 </li><li> mStrawberry OP9의 flasks에 공동 문화 차별 미디어 추가 </li><li> mStrawberry OP9 세포의 플라스크에 플레이트 1x10 3 ES 세포 / cm 2. 37 세포를 품어 ° C 5 % 습도 CO 2. </li></ol></li><li> mStrawberry OP9 전지 (주 2) <ol class="lalpha"><li> Subculture의 mStrawberry OP9가 그래서 당신은 공동 문화의 날 5 mStrawberry OP9 세포의 합류 레이어를 가지고. 주 5 confluency에 대한 1x10 4 / cm 2 판 mStrawberry은 OP9 세포 (단계 3.2 등). 일 9분의 8 필요한 경우 (같은 단계 2.2)에 대한 추가 subculture 세포를해야합니다. </li></ol></li><li> ES / mStrawberry OP9 공동 문화 (3 일) <ol class="lalpha"><li> 조심스럽게 공동 문화 매체를 제거하고 신선한 공동 문화 차별화 매체로 교체 </li></ol></li><li> ES / mStrawberry OP9 공동 문화 (5 일) <ol class="lalpha"><li> 조심스럽게 PBS로 2 배 공동 문화를 씻어 </li><li> 사전 예열 0.25 % 트립신 - EDTA (에틸렌 다이아 민 테트라 초산)과 세포 Trypsinize </li><li> 공동 문화 차별화 미디어 플라스크에서 세포를 씻어 </li><li> 400xg에서 펠렛 세포는, 7min, RT가. 뜨는 취소 </li><li>세포에 공동 문화 차별 미디어를 추가. 세포를 resuspend하고 단일 세포 현탁액에 대한 보장하기 위해 21 게이지 바늘을 사용 bluntended </li><li> hemocytometer를 사용하여 셀을 계산합니다. 주 0에서 ES 세포 번호 100-150 배 증가 있어야합니다. </li><li> 다시 씨앗 6.48x10 4 / cm 2 - 합류, mStrawberry OP9 세포의 새로운 레이어에 7.76x10 4 / cm 2 공동 문화 전지 </li><li> 일 5 ASSAYS - 남은 공동 문화 전지는 유동세포계측법 분석, cytopreps (30K 세포), 칩 assays 등을 사용할 수 있습니다 </li></ol></li><li> ES / mStrawberry OP9 공동 문화 (주 9분의 8 앤드 데이 12) <ol class="lalpha"><li> 플라스크에서 차별 미디어를 제거하고 저장합니다. 서스펜션의 조혈 세포를 제거하는 PBS로 세척 monolayer 1X가. 조혈 세포를 밖으로 던져하지 마십시오. </li><li> 사전 예열 0.25 % 트립신 - EDTA (에틸렌 다이아 민 테트라 초산)와 자기편 세포 Trypsinize </li><li> ES의 차별화 미디어 플라스크에서 세포를 씻고 정지 = 발견 조혈 세포에 추가 </li><li> 400xg에서 펠렛 세포는, 7min, RT가. 뜨는 취소 </li><li> 세포에 공동의 문화 분화 미디어 또는 PBS를 추가합니다. 셀을 resuspend하고 hemocytometer를 사용하여 단일 세포 현탁액의 개수 세포에 대한 보장하기 위해 21 게이지 바늘 bluntended을 사용합니다. </li><li> 공동 문화 일 12 일에 다시 다음 주 8 9 합류, mStrawberry OP9 세포의 새 레이어에 다시 시딩 공동 문화 세포에 의해 더 성숙 조혈 progenitors을 평가하고 14 일 동안 계속하실 수 있습니다. 분석의 하루 필요한 세포 회복에 따라 다시 씨앗 최적의 핸드폰 번호. </li></ol></li><li> ES - 파생 자손 (주 1-14)의 평가. ES - 파생 자손은 mStrawberry 부정적인 세포 (ES - 파생 자손) 밖으로 정렬 흐름 공동 문화의 모든 일에 평가할 수있다. <ol class="lalpha"><li> 플라스크에서 차별 미디어를 제거하고 저장합니다. PBS로 세척 monolayer 1X는 정지 조혈 세포를 제거하려면 다음과 같이하십시오. 조혈 세포를 밖으로 던져하지 마십시오. </li><li> 사전 예열 0.25 % 트립신 - EDTA (에틸렌 다이아 민 테트라 초산)와 자기편 세포 Trypsinize </li><li> 공동 문화 차별화 미디어 플라스크에서 세포를 씻고 정지에있는 조혈 세포에 추가 </li><li> 400xg에서 펠렛 세포, 7min, RT. 뜨는 취소 </li><li> 21 게이지 바늘 bluntended 및 계산과 셀 Resuspend </li><li> ES - 파생 자손 (mStrawberry 부정적인 세포)는 mStrawberry 긍정적인 OP9 세포에서 정렬 흐름하실 수 있습니다. ES - 파생 자손은 다음 유동세포계측법 분석, cytopreps, 칩 assays 등 특색 수 있습니다 </li></ol></li></ol> 4. 대표 결과 : <img src="/files/ftp_upload/2559/2559fig1.jpg" alt="그림 1" /> 그림 1. ES / mStrawberry OP9 공동 문화 시스템. 우리 연구실 모델 조혈 개발에 체외 ES 세포 공동 배양 시스템을 사용합니다. mStrawberry OP9 세포의 합류 레이어에 배치 때이 공동 문화 시스템에서 ES 세포는 Day5에서 hemangioblasts로, 차별. 말기 차별화된 조혈 lineages은 주 14 나타나는 동안 공동 문화가 진행되는 동안, 조혈 성의 전구 물질은 8 일 현재입니다. 필요에 mStrawberry OP9 세포의 새로운 레이어에 합류 ES - 파생 자손의 passaging 때 화살표가 차별화의 일 나타냅니다. <img src="/files/ftp_upload/2559/2559fig2.jpg" alt="그림 2" /> 그림 2. 적절한 ES 세포 차별화. ES 세포는 일 0 mStrawberry OP9의 합류 계층에 씨앗을 품고있다. ES - 파생 자손은 ES - 파생 자손이 정의된 테두리와 셀 클러스터처럼 보이는 또는 소용 돌 이꼴 패턴을 형성하기 시작과 분화의 날 3 표시되기 시작합니다. 5 일째에 세포의 whorls을 쌓아으로 내피와 조혈 lineages에 대한 일반적인 mesodermal 전구체 아르 Hemangioblasts는 나타납니다. 오일 이상 공동 문화 기간 동안, ES - 유래 자손은 조혈 세포의 클러스터로 나타납니다. 두 시간 내지 네 단세포 클러스터, 6 일 7 /, 나타납니다 및 공동 문화의 날 9분의 8로 큰 휴대 클러스터로 성장합니다. 기타 ES 파생 자손도 단단히 자기편 stromal 세포 레이어 바운드 볼 수 있습니다. <img src="/files/ftp_upload/2559/2559fig3.jpg" alt="그림 3" /> 그림 3. 이 공동의 문화에서 실패한 ES 세포 차별화. 결과 부적 절한 ES 세포의 차별을 반영합니다. ES - 파생 자손은 5 일에 소용 돌 이꼴 무늬에 나타나지 어떻게 있습니다. 소용 돌 이꼴 무늬에 세포의 부재는 부적 절한 hemangioblast 형성을 나타냅니다. hemangioblast이 제대로 개발하지 않으면, 다음 공동 문화 저하 조혈성 혈통 차별집니다 계속했다. 부적 절한 hemangioblast 형성 공동 문화가 폐기해야합니다. <img src="/files/ftp_upload/2559/2559fig4.jpg" alt="그림 4" /> 그림 4. mStrawberry OP9 세포 문화. (A) mStrawberry OP9 세포가에 subcultured 아르confluency 70~90%. (B) 적절한 차별 화를위한, ES 세포와 ES - 파생 자손은 mStrawberry OP9 세포 지나치게 합류 계층에 배치됩니다. <img src="/files/ftp_upload/2559/2559fig5.jpg" alt="그림 5" /> 그림 5. 대표 ES / mStrawberry OP9 공동 문화 흐름 프로필. 모든 ES - 파생 자손은 mStrawberry 부정적인 세포이고 mStrawberry OP9 세포에서 정렬 흐름하실 수 있습니다. mStrawberry 부정적인 세포 게이트는 전통적인 ES/OP9 공동 문화를 사용하여 결정되었다.

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관심 없음 충돌 선언하지 않습니다.

공동 문화의 날 5 Flk1 이상 hemangioblasts의 생산에 mStrawberry OP9 세포 결과 합류 레이어에 ES 세포의 적절한 분화. Hemangioblasts는 그림 1에서 관찰된 세포의 whorls을 쌓여 나타납니다. 공동 문화의 나머지 내용은 보이지 ES - 파생 자손은 조혈 세포의 클러스터 (그림 1)과 같이 나타납니다. Day6 / 7 2-4 셀 클러스터가 나타납니다 및 공동 문화의 날 9분의 8로 큰 세포 클러스터 (자기편과 정지에 모두)로 성장합니다. 기타 ES 파생 자손도 단단히 자기편 stromal 세포 계층 내에서 바운드 볼 수 있습니다. 프로토콜에 표시되지 않지만, 그것은 FBS가 ES 성장 매체, mStrawberry OP9 성장 매체와 ES / mStrawberry OP9 차별화 미디어에서 사용되는 미리 테스트하는 것이 중요합니다. 제대로 혈청 분화의 5 일 전까지 공동 문화에 적절한 mStrawberry OP9 세포의 성장뿐만 아니라, ES 세포의 적절한 분화를 보장해야 테스트. 대안 또는 mStrawberry OP9 세포 이외에, OP9 전지 전에 (7.8x10 4 셀 / cm 2) confluency에서 시딩하기 위해 8000 래드에서 조사 수 있습니다. OP9 세포의 조사는 하류 assays에서 OP9 세포를 오염, 이전의 거의 완전한 제거를 허용뿐만 아니라, 반복 replating 단계의 제거를 허용합니다. OP9 세포가 CMV 프로 모터의 제어 (pRRLsin - CMV 벡터, UCLA 벡터 코어) 아래 lentiviral 벡터 표현 mStrawberry의 3ug/mL와 OP9 세포 transducing하여 mStrawberry 단백질을 표현할 수 있도록 설계되었습니다. 표준 ES/OP9 공동 문화 프로토콜은 세포 통로에서 OP9 세포를 오염 오래된 줄이기 위해뿐만 아니라, 합류 레이어 OP9 세포에 5 일 replating 단계를 ES 세포의 위치를​​ 수반. 또한, 정지에서 발견 유일한 조혈 세포 ES - 파생 자손의 평가를 위해 제거됩니다. 그러나, replating 단계만을 정지 세포의 수확 모두 하나가 잠재적인 개발의 상당수 ES - 파생 조혈 세포를 그리워. 이 문제는 녹아웃 에스 라인과 관련된 조혈 결함을 특성화하려고 할 때 주소로 중요 해지고 있습니다. 이 문제를 우회하기 위해 우리 실험실 OP9 세포를 mStrawberry - 표현의 사용을 통해, 수정 공동 문화를 사용합니다. 이것은 모든 ES의 자손은 매일 5 시에 공동 문화의 지속으로 전송됩니다 것을 보장하고, 하류 assays 모든 ES - 파생 자손의 수확. 이 수정은 인간 및 마우스의 ES 라인 모두에서 파생된 조혈 자손의 평가와 특성화에 유용한 도구를 제공합니다. 다양한 단계와 최종 원시 조혈이 ES - mStrawberry OP9 공동 문화 모델 recapitulates. 조혈 개발의 초기 단계를 공부에 많은 사람들이 ES 세포에서 hemangioblast의 발전을 평가 BL - CFC (콜로니 형성 세포 블래스트와 같은) 분석을 사용합니다. 초기 조혈 개발을 조사 때 BL - CFC 분석이 유용한 도구이지만 그것이 hemangioblast의 평가뿐만 아니라, 더 많은 성인 조혈 lineages을 허용으로 mStrawberry - ES 공동 문화 시스템은 증가 유틸리티를 전시하고있다. 전통 OP9/ES 및 EB 차별 프로토콜을 사용하여 이전 작업 순서 등 HoxB4의 overexpression로, 아마도 필요한 추가 요소가 체외에서 조혈 줄기 세포를 repopulating 장기 파생 것으로 나타났습니다. 추가 작업은 mStrawberry 부정, 정렬 ES - 파생 인구 사실 조혈 줄기 세포를 포함 여부를 평가하는 것이 필요합니다.

OP - 9 셀 유지 보수 중 스톡 구성 요소 <table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> aMEM </td><td> Invitrogen </td><td> 12571-063 </td><td></td></tr><tr><td> FBS </td><td> 오메가 과학 </td><td> FB01 </td><td> 사전 테스트 </td></tr><tr><td> L - 글루타민 </td><td> Cellgro </td><td> 25-005 - CI </td><td> 200nM </td></tr><tr><td> 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) </td><td> 세포 기술을 줄기 </td><td> 07,901 </td><td></td></tr></tbody></table> ES/OP-9 세포 차별화 미디엄 스톡 구성 요소 <table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> aMEM </td><td> Invitrogen </td><td> 12571-063 </td><td></td></tr><tr><td> FBS </td><td> Hyclone </td><td> SH30070 </td><td> 사전 테스트 </td></tr><tr><td> L - 글루타민 </td><td> Cellgro </td><td> 25-005 - CI </td><td> 200mM </td></tr><tr><td> PBS (CA이없는 +와 MG 2 +) </td><td> Cellgro </td><td> 21-031 - CV </td><td></td></tr><tr><td> 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) </td><td> 세포 기술을 줄기 </td><td> 07,901 </td><td></td></tr></tbody></table>

modified es / op9 co-culture protocol provides enhanced characterization of hematopoietic progeny

생체내 및 배아 lethalities 처리하지 않고, 조혈에 관련된 최초의 유전자를 특성화에 대한 액세스하기 어렵습니다 세포 인구를 공부하면 조혈 세포의 운명으로 ES 세포의 체외에서 차별에 유용합니다. OP9 stromal 세포 라인 M - CSF 기능 부족 osteopetrosis 돌연변이 생쥐의 calvaria에서 파생되는 등 ES/OP9 공동 문화 시스템은 원래, macrophages의 이상 생산하지 않고, 조혈 자손을 생산하도록 설계되었습니다. 체외 ES/OP9 공동 문화 시스템은 초기 조혈 발전을 요점을 되풀이하기 위해 사용할 수 있습니다. OP9 stromal 세포에 교양하면, ES 세포는 Flk - 1 + hemangioblasts, 조혈 progenitors, 마침내 성숙, 말기 차별 lineages로 구분. 표준 ES/OP9 공동 문화 프로토콜 OP9 세포의 합류 계층에 ES 세포의 위치를​​ 알아볼 수, OP9 세포를 오염 오래된 제거하기 위해뿐만 아니라, 정기 replating 단계를 반복합니다. 또한, 현재의 프로토콜은 정지에서 발견은 조혈 세포를 평가 참여하고 분화의 각 하루 ES - 파생 자손의 평가에 최적화된되지 않습니다. 그러나, replating 단계만을 정지 세포의 수확 하나의 가능성이 ES - 파생 자손과 조혈 세포를 개발의 큰 부분을 그리워. 이 문제는 녹아웃 에스 라인과 관련된 조혈 결함을 특성화하려고 할 때 주소로 중요 해지고 있습니다. 여기 하류 assays에서 OP9 세포 오염의 제거를 허용 수정 ES / mStrawberry OP9 공동 문화를 설명합니다. 이 방법은 더 직접적으로 배아 내에서 관찰 조혈성 차별화 패턴에 해당하는 조혈 차별화 패턴의 결과로, 공동 문화의 모든 일에 모든 ES - 파생 자손의 전체 평가 수 있습니다.

Watch this Scientific Journal Video about Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny at JoVE.com

Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

mStrawberry OP9 세포가 공동 문화의 모든 ES - 파생 자손의 완전한 평가를 위해 수 있습니다.

수정 ES / OP9 공동 문화 프로토콜 조혈 제니의 향상된 특성화를 제공합니다

The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo ...

modified-es-op9-co-culture-protocol-provides-enhanced

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JoVE Journal

Biology

20.6K 조회수.  University of California, Los Angeles. mStrawberry OP9 세포가 공동 문화의 모든 ES - 파생 자손의 완전한 평가를 위해 수 있습니다.

비디오: Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

Freezing Human ES Cells

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction.  Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature.  During this time cells can be observed rounding, and colonies lifting off the plate surface.  Gentle repeated pipetting will remove cells and colonies from the plate surface.  Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun.  Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10cm plate.  After cells are adequately resuspended, ice cold freezing media is added at equal volume.  Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80C over 24 hours.  Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.

냉동 인간 ES 세포

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove ...

연구

생물학

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

냉동 세포에서 시작 문화 : 인간 ES 세포

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

ES Cell-derived Neuroepithelial Cell Cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

ES 세포 - 파생 Neuroepithelial 셀 문화

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

인간의 배아 줄기 (ES) 세포의 전파

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Neurotoxicity 높은 컨텐츠 분석의 연결 및 Astrocyte 공동 문화 분석

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

다중 구획 CNS 뉴런 - glia 공동 문화 Microfluidic 플랫폼

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

Microfluidic 가용성식이 중재 신호의 상호 작용을 조사하는 상피 세포 박테리아의 공동 문화

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

세 가지 차원 혼합 셀 회전 타원체 공동 문화에서 Mammary 상피 세포의 분리

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. Deviations from what is considered a healthy microbiota composition (dysbiosis) may impair vital functions leading to pathologic conditions. Recent and ongoing research efforts have been directed toward the characterization of associations between microbial composition and human health and disease.
Advances in high-throughput sequencing technologies enable characterization of the gut microbial composition. These methods include 16S rRNA-amplicon sequencing and shotgun sequencing. 16S rRNA-amplicon sequencing is used to profile taxonomical composition, while shotgun sequencing provides additional information about gene predictions and functional annotation. An advantage in using a targeted sequencing method of the 16S rRNA gene variable region is its substantially lower cost compared to shotgun sequencing. Sequence differences in the 16S rRNA gene are used as a microbial fingerprint to identify and quantify different taxa within an individual sample.
Major international efforts have enlisted standards for 16S rRNA-amplicon sequencing. However, several studies report a common source of variation caused by batch effect. To minimize this effect, uniformed protocols for sample collection, processing, and sequencing must be implemented. This protocol proposes the integration of broadly used protocols starting from fecal sample collection to data analyses. This protocol includes a column-free, direct-PCR approach that enables simultaneous handling and DNA extraction of large numbers of fecal samples, along with PCR amplification of the V4 region. In addition, the protocol describes the analysis pipeline and provides a script using the latest version of QIIME (QIIME 2 version 2017.7.0 and DADA2). This step-by-step protocol is aimed to guide those interested in initiating the use of 16S rRNA-amplicon sequencing in a robust, reproductive, easy to use, detailed way.

This manuscript describes a detailed standardized protocol of high-throughput 16S rRNA-amplicon sequencing. The protocol introduces an integrated, uniformed, feasible, and inexpensive protocol starting from fecal sample collection through data analyses. This protocol enables analysis of large numbers of samples with rigorous standards and several controls.

16S rRNA Amplicon 시퀀싱에 의해 배설물 미생물 특성화에 대 한 가이드 프로토콜

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. ...

Development of a Cell Co-Culture Model to Mimic Cardiac Ischemia/Reperfusion In Vitro

Ischemic heart disease is the leading cause of death and disability worldwide. Reperfusion causes additional injury beyond ischemia. Endothelial cells (ECs) can protect cardiomyocytes (CMs) from reperfusion injury through cell-cell interactions. Co-cultures can help investigate the role of cell-cell interactions. A mixed co-culture is the simplest approach but is limited as isolated treatments and downstream analyses of single cell types are not feasible. To investigate whether ECs can dose-dependently attenuate CM cell damage and whether this protection can be further optimized by varying the contact distance between the two cell lines, we used Mouse Primary Coronary Artery Endothelial Cells and Adult Mouse Cardiomyocytes to test three types of cell culture inserts which varied in their inter-cell layer distance at 0.5, 1.0, and 2.0 mm, respectively. In CMs-only, cellular injury as assessed by lactate dehydrogenase (LDH) release increased significantly during hypoxia and further upon reoxygenation when the distance was 2.0 mm compared to 0.5 and 1.0 mm. When ECs and CMs were in nearly direct contact (0.5 mm), there was only a mild attenuation of the reoxygenation injury of CMs following hypoxia. This attenuation was significantly increased when the spatial distance was 1.0 mm. With 2.0 mm distance, ECs attenuated CM injury during both hypoxia and hypoxia/reoxygenation, indicating that sufficient culture distancing is necessary for ECs to crosstalk with CMs, so that secreted signal molecules can circulate and fully stimulate protective pathways. Our findings suggest, for the first time, that optimizing the EC/CM co-culture spatial environment is necessary to provide a favorable in vitro model for testing the role of ECs in CM-protection against simulated ischemia/reperfusion injury. The goal of this report is to provide a step-by-step approach for investigators to use this important model to their advantage.

Development of a Cell Co-Culture Model to Mimic Cardiac Ischemia/Reperfusion In Vitro

Spatial distance is a key parameter in assessing hypoxia/reoxygenation injury in a co-culture model of separate endothelial and cardiomyocyte cell layers, suggesting, for the first time, that optimizing the co-culture spatial environment is necessary to provide a favorable in vitro model for testing the role of endothelial cells in cardiomyocyte protection.

체외에서 심장 허혈/재관류를 모방하기 위한 세포 공동 배양 모델 개발

Ischemic heart disease is the leading cause of death and disability worldwide. Reperfusion causes additional injury beyond ischemia. Endothelial cells ...