Name: feeder-free adaptation, culture and passaging of human ips cells using complete knockout serum replacement feeder-free medium
Uploaded: 2010-07-15T17:00:00.000+00:00
Duration: 8 min 29 s
Description: Watch this Scientific Journal Video about Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium at JoVE.com

참고 : 무균 문화 조건을 유지하기 위해서는이 프로토콜의 절차를 모두 무균 실험실 방법을 사용하여 수행하고 층류 후드 아래에 실시하고 있습니다. 시작하기 전에, 모든 미디어는 37 equilibrated되도록 ° C 적절하게 기름. Geltrex - 코팅 문화 요리를 준비 참고 : CELLstart 코팅 문화 요리의 사용을 위해 부록을 참조하십시오 <ol><li> 해동 한 천천히 2-8에서 Geltrex (1 ML) ° C의 튜브 및 넉아웃 D-MEM/F-12 99 ML에 1:100 그것을 희석. 부드럽게 솔루션을 섞는다. 참고 : 일부 IPS 세포 라인은 최적의 성장을 위해 다른 Geltrex 희석이 필요할 수 있습니다. 대체 dilutions에 대한 부록을 참조하십시오. </li><li> Geltrex 솔루션 (35 - mm 요리 1 ML, 60 mm 요리 1.5 ML) 각 문화 요리의 전체 표면을 커버. </li><li> 건조를 방지하고 37에서 1 시간 동안 요리를 부화 Parafilm 각 접시 ° C. 봉쇄해 참고 : 최대 1 개월이 지점에서 4 ° C에서 Geltrex - 코팅 문화 요리를 저장할 수 있습니다. 밖으로 건조에서 Geltrex을 방지하기 위해 Parafilm 각 요리를 밀봉합니다. </li><li> 이전 사용하기 층류 후드에 Geltrex - 코팅 요리를 전송하고는 실온 (약 1 시간)에 평형 수 있습니다. </li></ol> 완료 녹아웃의 SR 공급기 - 무료 매체를 준비 <ol><li> 10 μg / ML 기본 FGF 솔루션 1 ML를 준비하려면, 무균 아래에 나열된 구성 요소를 섞는다. -20 ° C에서 나누어지는 솔루션 및 저장 최대 6 개월까지. <table border="0"><tbody><tr><td> 기본 FGF </td><td> 10 μg </td></tr><tr><td> D - PBS </td><td> 990 μL </td></tr><tr><td> 10% BSA </td><td> 10 μL </td></tr></tbody></table> 참고 : BSA는 같은 농도에서 HSA 또는 넉아웃의 SR로 대체 수 있습니다. </li><li> 2 MG / ML Dispase 솔루션의 50 ML를 준비하려면, 무균 아래에 나열된 구성 요소를 섞는다. 4 솔루션 및 저장 ° C 최대 2 주 동안을 소독 필터. <table border="0"><tbody><tr><td> Dispase </td><td> 100 MG </td></tr><tr><td> D - PBS </td><td> 50 ML </td></tr></tbody></table></li><li> 전체 녹아웃의 SR 피더 - 무료 (KSR - FF) 매체 무균 아래 표에 나와있는 구성 요소를 결합 100 ML를 준비합니다. <table border="1"><tbody><tr><td> 구성 요소 </td><td> 주식 농도 </td><td> 최종 농도 </td><td> 음량 </td></tr><tr><td> 마네 DMEM/F12 (Cat. 번호. 12660-012) </td><td> - </td><td> 1X </td><td> 76.8 ML </td></tr><tr><td> GlutaMAX - I (Cat. 번호 35050-061) </td><td> 200 MM </td><td> 2 MM </td><td> 1 ML </td></tr><tr><td> 녹아웃 SR (Cat. 번호. 10828-028) </td><td> - </td><td> 20% </td><td> 20 ML </td></tr><tr><td> 녹아웃 SR - GFC (Cat. 번호. A10580 - 01) </td><td> 50X </td><td> 1X </td><td> 2 ML </td></tr><tr><td> bFGF (Cat. 번호. PHG0024) </td><td> 10 μg / ML </td><td> 20 NG / ML </td><td> 200 μL </td></tr></tbody></table> 최대 일주일을 위해 당신은 2-8 ° C에서 KSR - FF 매체를 저장할 수 있습니다. </li><li> 온도와 가스의 전체 매체를 미리 equilibrating 바로 전에 무균 0.1 MM 최종 농도 2 메르 캅 토 에탄올의 필요한 볼륨 (55 MM 주식 농도)을 추가합니다. 예를 들어, KSR - FF 매체의 100 ML를 준비하는 것은 2 - 메르 캅 토 에탄올 (1:550 희석) 또는 55 mm의 182 μL를 추가, 2 - 메르 캅 토 에탄올이에 대한 1X 완료 매체에 추가 2-8 ° C에서 저장될 수 있습니다 최대 일주일. </li></ol> KSR - FF 중간에 iPSCs를 적응 <ol><li> 시작하기 전에, 후드에서 상온에 Geltrex 코팅 요리를 미리 평형. </li><li> 문화 MEF 피더 세포에서 iPSCs 그들은 70-80%의 합류 때까지. MEF 기반 문화 프로토콜에 대한 GIBCO 마우스 배아의 Fibroblast 수첩을 참조하십시오. </리&gt; <li> 37 ° C의 물을 욕조에 Dispase 사전 따뜻한 필요한 볼륨. 필요한 볼륨에 대한 자세한 내용은 아래의 표 1을 참조하십시오. </li><li> 37 ° C의 물 목욕 for15 분에 KSR - FF의 필요한 볼륨을 사전에이 - 평형. 필요한 볼륨에 대한 자세한 내용은 아래의 표 1을 참조하십시오. </li><li> 문화 요리에서 매체를 기음과 Dispase 솔루션의 적절한 금액을 추가합니다. 3~5분에 대해 37 ° C에서 요리를 품다. </li><li> 각 문화 선박에서 Dispase 솔루션을 기음과 D - PBS (2 3 회)로 부드럽게 MEF의 피더 세포를 씻어. </li><li> 각 문화 선박을 완료 마네의 SR 매체의 적절한 금액을 추가합니다. 부드럽게 문화 선박의 표면에서 세포를 긁어 셀 스크레이퍼 또는 5 ML의 피펫을 사용합니다. 참고 : 하드 IP를 적응 세포 라인에 대한 대체 적응 프로토콜에 대한 부록을 참조하십시오, </li><li> 별도의 15 ML 원뿔 튜브에 각 문화 접시에서 세포 현탁액를 수집합니다. 전체 녹아웃의 SR 매체의 적절한 금액을 각 문화 선박 린스, 그리고 D - PBS는 세포 현탁액을 포함 15 ML 원뿔 튜브로 매체를 씻어 추가합니다. 하나의 세포로 세포 대단히 짧은 시간을 휴식하지 않는주의 </li><li> 펠렛 iPSCs 5 분 200 XG에 15 ML 원뿔 튜브를 원심 분리기. </li><li> iPSC 펠렛에서 뜨는을 대기음. 분할 비율 (표 1)에 따라 KSR - FF 매체의 적절한 금액 펠렛을 Resuspend. 작은 대단히 짧은 시간이 표면에 잘 부착하지 않기 때문에, 작은 크기로 휴대 대단히 짧은 시간을 아프게하지 마십시오. 참고 : iPSCs이 KSR - FF 매체에 iPSC MEF 문화 미디엄에서 직접 passaged 후에 우리는 처음 3 구절에 대한 1시 2분의 분할 비율을 권장합니다. 일반적으로 1시 3분과 1시 5분 사이의 분할 비율이 적합하지만, 1시 2분에서 passaging 것은 피더 무료 문화에 적응 할 때 필요한 세포의 높은 밀도를 보장합니다. </li><li> 이전 Geltrex 코팅 요리 iPSCs을 도금하는 사전 코팅 접시에서 잔여 Geltrex 솔루션을 기음, 천천히 각 문화 요리에 세포 현탁액의 적절한 금액을 추가합니다. 참고 : 도금하기 전에 요리를 씻어하지 않습니다. </li><li> 접시의 표면에 걸쳐 세포를 해산하는쪽으로 여러 번 앞뒤로 측면 문화 요리를 이동합니다. 부드럽게 공기 4-6%의 CO 2의 humidified 분위기 37 ° C 배양기에서 배양 접시를 놓습니다. KSR - FF로 보낸 매체 매일 교체합니다. </li></ol> KSR - FF을 사용하여 Passaging 인간의 IPS 세포 <ol start="13"><li> 세포 70-80% 합류하고 subcultured 준비가되었는지 확인하기 위해 현미경으로 전체 KSR - FF의 성장 인간 iPSCs을 준수. 1 그림을 참조하십시오. 참고 : 식민지가 너무 밀도 또는 너무 커질 경우, 증가 차별이 발생합니다. </li><li> 잘라와 문화를 passaging하기 전에 어떠한 차별 iPSC 식민지를 제거합니다. </li><li> 37 ° C의 물을 욕조에 Dispase 사전 따뜻한 필요한 볼륨. 필요한 볼륨에 대한 자세한 내용은 아래의 표 1을 참조하십시오. </li><li> 37 ° C의 물 목욕 for15 분에 KSR - FF의 필요한 볼륨을 사전에이 - 평형. 필요한 볼륨에 대한 자세한 내용은 아래의 표 1을 참조하십시오. </li><li> 피펫을 사용하여 문화 선박에서 보낸 매체를 대기음하고, D - PBS로 두 번 세포를 씻어. </li><li> 부드럽게 문화 선박 (예, 60 mm 문화 요리 당 Dispase 솔루션 1 ML)로 사전 예열 Dispase 솔루션을 추가합니다. 코트 전체 세포 표면에 소용돌이 문화 우주선합니다. </li><li> 3 분 37 ° C에서 문화 함선을 품어. </li><li> 인큐베이터에서 혈관을 제거 Dispase 솔루션을 기음, 부드럽게 D - PBS로 세포를 씻어. </li><li> 부드럽게 세포 스크레이퍼를 사용하여 문화 접시의 표면에서 세포를 긁어 및 살균 15mL 원심 튜브에 세포를 전송합니다. </li><li> 부드럽게 분리하지 않은 세포를 &quot;해제 분사.&quot;, KSR - FF로 두 번 문화 접시를 씻어 수영장은 15mL 튜브에 세포와 매체를 씻어. </li><li> 상온에서 펠릿 세포 5 분 200 XG에서 튜브를 원심 분리기. </li><li> 조심스럽게 세포 펠렛을 방해하지 않고 표면에 뜨는를 대기음하고 폐기. </li><li> 부드럽게 튜브가 완전히 튜브 바닥에서 세포 펠렛을 이동시키다을 버려. </li><li> 부드럽게 5mL serological 피펫을 사용하여 사전 equilibrated KSR - FF의 세포를 resuspend. 씹다하지 마십시오. 참고 : 부드럽게 손상을 방지하기 위해 무력을 사용하지 않고 세포를 resuspend하는 단계에서 중요한 그것입니다. </li><li> 원하는 분할 비율에 신선한 60 mm Geltrex - 코팅 접시에 세포를 전송하고 표면을 가로질러 세포를 해산하는쪽으로 여러 번 앞뒤로 측면 문화 요리 이동합니다. </li><li> 의 humidified 분위기 37 ° C 배양기에서 배양 접시를 플레이스4~6%는 공중에 2 CO. </li><li> 그 다음날, 부드럽게 세포 파편을 제거하는 KSR - FF로 보낸 매체를 대체합니다. 이후 매일 지출 매체를 교체합니다. IPS 세포는 매일 그들로 (약 매 4~5일) 필요한 통로 관찰. Passaging은 세포가 70~80%의 합류에 도달했을 때 권장합니다. IPS 세포 cryopreservation 융해 들어, &quot;완료 마네 세럼 교체 피더 - 무료 매체를 사용하여 Cryopreserving 인적 IPS 세포 복구하기&quot;라는 제목의 프로토콜을 참조하십시오. </li></ol> 예상 결과 <img src="/files/ftp_upload/2236/2236fig1.jpg" alt="그림 1" /> 그림 1. 아래의 위상 콘트라스트 이미지는 완전 넉아웃 세럼 교체 피더 - 무료 매체를 사용하여 Geltrex - 코팅 문화 요리에 성장 iPSCs를 보여줍니다. iPSCs 큰 핵 및 세포질 부족한 특징 hESCs에 형태학 유사한 나타냅니다. (40x 확대) <table border="1"><tbody><tr><td> 구성 요소 </td><td> 35mm 디시 </td><td> 60mm 디시 </td><td> 100mm 디시 </td></tr><tr valign="top"><td> 완료 녹아웃 SR 미디어 </td><td> 2 ML </td><td> 4 ML </td><td> 10 ML </td></tr><tr valign="top"><td> Geltrex 솔루션 </td><td> 1 ML </td><td> 1.5 ML </td><td> 4-5 ML </td></tr><tr valign="top"><td> Dispase </td><td> 0.5 ML </td><td> 1 ML </td><td> 3-4 ML </td></tr><tr valign="top"><td> rinsing을위한 D - PBS </td><td> 2 ML </td><td> 4 ML </td><td> 10 ML </td></tr></tbody></table> 표 1. 추천 볼륨 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/2236/2236appendix.pdf">부록을 참조하려면 여기를 클릭하십시오</a> .

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이 문서의 저자는이 문서에서 사용되는 시약 및 악기를 생산하는 생명 기술에 의해 고용됩니다.

<table><tbody><tr><td>Knockout DMEM/F12</td><td></td><td>Cat. no. 12660-012</td><td>Note: see appendix for the use of alternative DMEM products</td></tr><tr><td>GlutaMAX-I</td><td></td><td>Cat. No. 35050-061</td><td></td></tr><tr><td>KnockOut Serum Replacement</td><td></td><td>KnockOut SR, Cat. no. 10828-028</td><td></td></tr><tr><td>KnockOut Serum Replacement Growth Factor Cocktail</td><td></td><td>KnockOut SR-GFC, Cat. no. A10580-01</td><td></td></tr><tr><td>FGF-basic, human recombinant protein, 10 &amp;mu;g</td><td></td><td>bFGF, Cat. no. PHG0024</td><td>Note: see appendix for alternative bFGF pack sizes.</td></tr><tr><td>2-Mercapt&amp;#8211;thanol</td><td></td><td>Cat. no. 21985-023</td><td></td></tr><tr><td>Dispase</td><td></td><td>Cat. no. 17105-041</td><td>Note: see appendix for alternative passaging methods.</td></tr><tr><td>Geltrex hESC-Qualified Reduced Growth Factor Basement Matrix, 1 mL</td><td></td><td>Cat. no. A10480-01</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (D-PBS) without calcium and magnesium </td><td></td><td>Cat. no. 14190-144</td><td></td></tr><tr><td>Sterile Tissue Culture Hood</td><td></td><td></td><td></td></tr><tr><td>Incubator set at 37&amp;#x00B0;C</td><td></td><td></td><td></td></tr><tr><td>Pipette-Aid</td><td></td><td></td><td></td></tr><tr><td>Water Bath set at 37&amp;#x00B0;C</td><td></td><td></td><td></td></tr><tr><td>Sterile serological pipettes (5 mL, 10 mL)</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td></td><td></td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>60mm Tissue Culture treated dishes</td><td></td><td></td><td></td></tr></tbody></table>

feeder-free adaptation, culture and passaging of human ips cells using complete knockout serum replacement feeder-free medium

인간과 마우스 섬유아 세포가 배아 줄기 세포에 상당히 유사 자질을 가진 1-3 IPS 세포를 생성하는 재설정 될 수 있다고 2006 년에 발견은 약물 발견, 세포 치료와 기초 연구를위한 pluripotent 세포의 중요한 새로운 소스를 만들었습니다. GIBCO 미디어 및 시약 년 동안 pluripotent 줄기 세포 연구의 선두에있다. DMEM가 마네 세럼 교체와 보충 마네는 배아 줄기 세포의 성장과 지금은 IPS 세포 배양 3-9위한 최고의 매체입니다. 이 황금 표준 미디어 시스템은 이제 마네의 SR 성장 팩터 칵테일의 추가와 함께 피더 무료 문화 사용할 수 있습니다. 전통적인 인간의 ES와 IPS의 세포 배양 방법은 마우 스나 인간 fibroblast 피더 레이어의 사용 또는 피더 - 에어컨 매체를 필요로합니다. 이러한 문화 방법은 규모에 노동 집약, 하드 있으며, 그것은 정의되지 않은 상황으로 인해 undifferentiated 허리의 세포를 유지하기 어렵습니다. Invitrogen은 여전히​​ 마네의 SR의 사용을 유지하면서 당신이 피더 - 무료로 전환 엉덩이 셀 문화를 쉽게하도록 마네의 SR 성장 팩터 칵테일을 개발했습니다.

Watch this Scientific Journal Video about Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium at JoVE.com

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

다음 프로토콜은 녹아웃 완료 세럼 교체 피더 - 프리 미디어 (KSR - FF)를 사용하여 피더 무료 문화 인간 유도 Pluripotent 줄기 (IPS) 셀을 적응을위한 지침을 제공합니다. 일단 지속적인 유지 보수를위한 지침도 제공되며, 적응.

완료 녹아웃 세럼 교체 피더 - 무료 매체를 사용하여 인간의 IPS 세포 피더 - 자유 적응력, 문화와 Passaging

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

feeder-free-adaptation-culture-passaging-human-ips-cells-using

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

22.4K 조회수.  Life Technologies.  中文 日本語 

비디오: Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient specificity and characteristic stemness. However, the pluripotency of iPSCs, which is essential to their functionality, must be confirmed before these cells can be used in such applications. While a rigorous characterization of iPSCs, through different cellular and functional assays is necessary to establish their pluripotency, routine assessment of pluripotency maintenance can be achieved more simply and effectively through immunocytochemical techniques. Here, we present a systematic protocol for culturing hiPSCs, in a scaled-down manner, to particularly facilitate the verification of their pluripotent state using immunocytochemistry. More specifically, this methodology encompasses an efficient and cost-effective means of growing iPSCs in serum-free conditions and plating them on small chamber slides or glass coverslips ideal for immunocytochemistry.

Regular characterization of induced pluripotent stem cells (iPSCs), to ascertain maintenance of their pluripotent state, is an important step before these cells are used for other applications. Here we describe a method for the small-scale propagation of human iPSCs specifically designed to enable their easy and routine characterization via immunocytochemistry.

일상적인 면역 세포 화학 특성에 대한 혈청이없는 조건에서 인간 iPSCs의 소규모 전파

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient ...

연구

발생학

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

정의 된 이종 무료 및 급지대 무료 문화 세대의 인간 iPSC 파생 된 망막 세포 모델에 대 한

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

인간 유도에서 Somite 파생 상품의 체 외 발생에서 만능 줄기 세포

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

인간 다 능성에서 신경 능선 세포의 전구 세포의 공급이없는 유도는 줄기 세포

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

신경과학

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

Cryopreserving와 인간 IPS 세포 복구 완료 마네에게 세럼 교체 피더 - 무료 매체를 사용하여

생물학

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Culturing 인간의 배아와 유도 Pluripotent 줄기 세포에 적합 마우스 배아 Fibroblast 세포의 준비

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

인간의 Cardiomyocytes의 생성 : 피더없는 인간 유도 만능 줄기 세포에서 분화 프로토콜

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

에피 솜과 HDAC 억제제를 사용하여 혈액에서 효율적인 유도 만능 줄기 세포 생성

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Advances in Human Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-Expressing Natural Killer Cells

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of human induced pluripotent stem cell (iPSC)-derived chimeric antigen receptor (CAR) expressing NK cells has emerged as a promising avenue for "off the shelf" cancer immunotherapy. Here, we utilized an NK cell-optimized CAR construct that includes the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3&#950; signaling domain, which has been demonstrated to stimulate robust antigen-specific NK cell-mediated antitumor activity. The use of iPSCs for CAR NK cell generation offers several advantages, including homogenous CAR expression, scalability, reproducibility, and the potential for clinical application. This detailed step-by-step protocol from cell engineering to differentiation enables the generation of NK cell-optimized iPSC-derived CAR-expressing NK cells, providing a standardized and targeted cancer immunotherapy with improved antitumor activity and highlighting their potential as a promising treatment option for various malignancies.

Here, we present a method to differentiate and expand human iPSC-derived chimeric antigen receptor (CAR)-expressing natural killer cells with improved killing against various malignancies. This protocol demonstrates the differentiation and expansion of natural killer (NK) optimized iPSC-derived CAR-NK cells and the measurement of antitumor activity against various tumor cell lines.

인간 유도 만능 줄기 세포 유래 키메라 항원 수용체 발현 자연 살해 세포의 발전

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of ...

면역학 및 감염병학

Rapid, Cost-Efficient, Enzyme-Free Passaging of Human Pluripotent Stem Cells on Feeder Cells by Ethylenediaminetetraacetic Acid-Mediated Dis-Adhesion

Human pluripotent stem cells (human embryonic stem cells, hESCs, and human induced pluripotent stem cells, hiPSCs) were originally cultured on different types of feeder cells for maintenance in an undifferentiated state in long-term culture. This approach has been supplanted to a large extent by feeder-free culture protocols, but these involve more costly reagents and can promote a transition to a primed state, which restricts the cells&#39; differentiation capacity. In both feeder and feeder-free conditions, the harvesting of hESC or hiPSC colonies for passaging is a necessary procedure for expanding the cultures.
To provide an easy and high-yield procedure for passaging hESCs/hiPSCs cultured on feeder cells, we have established a harvesting method using dis-adhesion elicited by the calcium chelator ethylenediaminetetraacetic acid (EDTA). We have assessed the yield and quality of the resultant passaged cells by comparing this approach to the original mechanical harvesting approach, in which colonies are isolated with a scalpel under a microscope (mechanical harvesting was chosen as a comparator to avoid the reagent variability associated with enzymatic harvesting).
In one set of experiments, two different hESC lines were maintained on a feeder cell layer of human foreskin fibroblasts. Each line was subjected to multiple passages using EDTA-based or mechanical harvesting and assessed for&#160;colony size and morphology, cell density, stemness marker expression, differentiation to the three germ layers in embryoid bodies, and genomic aberrations. In another set of experiments, we used EDTA-based harvesting on two different hiPSC lines and obtained similar results. EDTA-induced dis-adhesion saved time and gave a higher yield of colonies of a more favorable size and more uniform morphology compared to mechanical harvesting. It was also faster than enzymatic harvesting and not prone to enzyme batch variability. The EDTA-induced dis-adhesion method also facilitates the transfer of hESC/hiPSC lines from feeder cell-based culture to feeder-free conditions if desired for downstream use and analysis.

To avoid the limitations associated with the enzymatic or mechanical passaging of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) cultured on feeder cells, we have established a fast, effective, cost-efficient, high-yield method for harvesting hESC or hiPSC colonies maintained on a feeder cell layer of human foreskin fibroblasts using EDTA-mediated dis-adhesion.

에틸렌디아민테트라아세트산 매개 탈접착에 의한 공급세포에서 인간 만능 줄기세포의 신속하고 비용 효율적이며 효소 없는 패스페이징

Human pluripotent stem cells (human embryonic stem cells, hESCs, and human induced pluripotent stem cells, hiPSCs) were originally cultured on different ...