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://www.jove.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.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Knockout DMEM/F12</td>
<td>&nbsp;</td>
<td>&nbsp;</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>&nbsp;</td>
<td>&nbsp;</td>
<td>Cat. No. 35050-061</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut Serum Replacement</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>KnockOut SR, Cat. no. 10828-028</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut Serum Replacement Growth Factor Cocktail</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>KnockOut SR-GFC, Cat. no. A10580-01</td>
<td>&nbsp;</td>
<tr>
<td>FGF-basic, human recombinant protein, 10 &mu;g</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>bFGF, Cat. no. PHG0024</td>
<td>Note: see appendix for alternative bFGF pack sizes.</td>
</tr>
<tr>
<td>2-Mercaptoethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Cat. no. 21985-023</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>&nbsp;</td>
<td>&nbsp;</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>&nbsp;</td>
<td>&nbsp;</td>
<td>Cat. no. A10480-01</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s Phosphate Buffered Saline (D-PBS) without calcium and magnesium </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Cat. no. 14190-144</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Tissue Culture Hood</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Incubator set at 37&deg;C</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pipette-Aid</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Water Bath set at 37&deg;C</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sterile serological pipettes (5 mL, 10 mL)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>15 mL centrifuge tubes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>60mm Tissue Culture treated dishes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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 ...

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

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

트립신과 Passaging 색채 인간의 배아 줄기 세포 - 라인

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

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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  ...

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

인간 신경 줄기 세포를 Passaging

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

인간 배아 줄기 세포의 문화 및 유지 관리

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

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

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 ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

인간 혈청에서 작은 비 암호화 RNA를의 분리

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

인간 iPS 세포의 마이크로 RNA 발현 프로파일, IP의 유래 망막 색소 상피 세포, 태아의 망막 색소 상피

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

포유류의 전체 배아 문화에 적합 쥐 혈청의 제조

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

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 ...