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View additional product information for Essential 8™ Adaptation Kit - FAQs (A25935)
63 product FAQs found
可以。在mTeSR培养基+BD Matrigel基底膜基质条件下培养并冻存的PSCs细胞可复苏至Gibco Essential 8培养基中并种植于VTN-N基质上。某些细胞系复苏至冻存前的生长培养基及基质中可能会效果更好。在随后的下一次传代过程中，就可使用EDTA将这些细胞传代至Gibco Essential 8培养基+VTN-N的组合中了。
有多株多能干细胞（PSC）系通过了Gibco Essential 8培养基的测试（Chen G, Gulbranson DR, Hou Z et al (2011) Chemically defined conditions for human iPSC derivation and culture.Nat Methods 8:424–429）。
bFGF对于细胞的存活与增殖至关重要。
我们测试和确认了适用于下列PSC生长培养基：Gibco Essential 8培养基，Gibco StemPro hESC SFM，mTesR1培养基和含有Gibco KnockOut血清替代物（KSR）的饲养层依赖的培养基。
可以，源自皮肤活检样本的成纤维细胞可在添加有EGF，凝血酶和皮质醇的Gibco Essential 8培养基中培养和增殖（Chen G, Gulbranson DR, Hou Z et al (2010) Chemically defined conditions for human iPSC derivation and culture.Nat Methods 8:424–429.）
胰岛素对于细胞的存活与增殖至关重要。
是的，细胞可以在完全 Gibco Essential 8 培养基和 10% DMSO 中进行常规冷冻。或者，即用型 CTS PSC Cryomedium（货号 A4238801）可提供更有效的解冻后恢复。
分散酶与胶原酶一类的酶并不适用于Gibco Essential 8培养基和Gibco玻连蛋白（VTN-N）的组合。使用这些酶类传代会导致细胞活力和贴壁能力下降。
培养在Gibco Essential 8培养基与VTN-N组合中的细胞应使用EDTA进行传代。
不可以。除传代的当天之外，细胞都应每天换液。
是的，ROCK 抑制剂可以与 Essential 8 培养基一起使用。我们建议使用 RevitaCell Supplement（货号 A2644501），它专门设计用于最大限度地减少压力对 PSC 的影响。
非常重要的是，Gibco Essential 8完全培养基需放置在室温下进行平衡，请勿在37°C水浴中平衡。bFGF活性会在从4°C至37°C的温度反复改变中迅速降低。
Gibco Essential 8完全培养基的保质期为2–8°C条件下放置两周。
我们建议在室温下解冻补充剂约 1 小时。
在配制Gibco Essential 8培养基时，不能使用其他货号的DMEM/F-12替代Gibco Essential 8基础培养基。试剂盒中提供的Gibco Essential 8基础培养基含有更高浓度的碳酸氢钠。
如需配置500 mL 完全Gibco Essential 8培养基，请将Gibco Essential 8添加剂（50X）室温化冻1小时，之后于无菌条件下将下列成份混合在一起：
Gibco Essential 8基础培养基：490 mL
Gibco Essential 8添加剂：10 mL
您预期可看到正常的多能干细胞（PSC）形态。PSC的预期生长形态是如图所示的典型形态为，包括边界清晰的紧密聚集克隆和高核质比。
培养于其它无饲养层培养体系的细胞，例如mTeSR培养基搭配Matrigel基底膜基质，或StemPro hESC SFM搭配Geltrex基质，也能够成功培养于Gibco Essential 8培养基搭配VTN-N的体系中。此外，在饲养层细胞搭配KnockOut SR中培养的PSC经证明也能够培养于Gibco Essential 8培养基搭配VTN-N的组合中。不过在更换培养基体系之前，细胞必须手工传代或用EDTA进行传代到Gibco Essential 8培养基搭配VTN-N的体系中。
相比现有其他种类的无饲养层培养基，Gibco Essential 8培养基的变异度更小。不同于其他含有超过20种高度变异性成份的培养基，Gibco Essential 8培养基在cGMP标准下生产，拥有最优化的配方和生长因子水平，能够以最小的变异度帮助用户获得最大的细胞健康度，多能性和生长效果。
Gibco Essential 8培养基与玻连蛋白经证明能够支持PSC的生长达50代以上而不会发生任何核型异常，同时这些PSC仍保持着分化为三胚层的能力。James Thomson实验室Chen等报道（Chen G, Gulbranson DR, Hou Z et al (2010) Chemically defined conditions for human iPSC derivation and culture.Nat Methods 8:424–429.），玻连蛋白的VTN-N变异体与Gibco Essential 8培养基联用时相比野生型玻连蛋白能更好地支持人多能干细胞的贴壁和存活。。
是的。Gibco Essential 8培养基不含异源成份，是仅有8种成份的培养基，能够为您提供健康和可靠的培养效果。
以Gibco玻连蛋白（VTN-N）作为细胞生长基质使用Gibco Essential 8培养基的条件相比使用其他无饲养层系统而言，有三个主要差别需要加以考量：
•通常细胞的传代时间比其他无饲养层培养基提前24小时左右。
•细胞在达到85%左右的汇合度时即需要传代。如果细胞传代时超过了85%的汇合度，细胞健康度及最终的细胞得率都会下降。
•细胞应使用EDTA进行传代。不推荐使用胶原酶或分散酶。
是的。Gibco Essential 8培养基中含有100 ng/mL碱性成纤维细胞生长因子（bFGF），因此无需额外添加bFGF。
Gibco Essential 8培养基仅含培养PSC所需的八种必需成份。本品最初由Chen等（Chen G, Gulbranson DR, Hou Z et al (2010) Chemically defined conditions for human iPSC derivation and culture.Nat Methods 8:424–429).研发， 目的是为了克服mTeSR培养基中所发现的变异度问题。Gibco Essential 8培养基中专门以不含白蛋白（BSA）的有限成份的设计减少变异度。Gibco Essential 8培养基为用户准备了方便的两组分试剂盒：500 mL Gibco Essential 8基础培养基与10 mL Gibco Essential 8添加剂（50X）。
Gibco Essential 8培养基是一种不含异源成份，无需饲养层的培养基产品，专为人源多能干细胞（PSC）的生长和增殖而配制。Gibco Essential 8培养基最初由James Thomson实验室的Chen等（Chen G, Gulbranson DR, Hou Z et al (2010) Chemically defined conditions for human iPSC derivation and culture.Nat Methods 8:424–429）开发，并通过Cellular Dynamics International验证；到目前为止已久经测试，可维持多类PSC细胞系的多能性。
iPSC-derived neurons have been cultured successfully on Poly-D-Lysine with a secondary coating of mouse and human laminin.
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We have evaluated the use of Cytotune-iPS 2.0 Sendai Reprogramming Kit (Cat. No. A16517) for somatic cell reprogramming of CD34+ blood cells. For CD34+ cells, follow the instructions provided in the Cytotune 2.0 reprogramming manual for feeder-free reprogramming (pgs. 39-44). On Day 3, you can utilize rhVTN-N (Cat. No. A14700), Geltrex (Cat. No. A1413302), or rhLaminin-521 (Cat. No. A29248 or A29249). From Day 8 onward, rather than feeding daily with Essential 8 Medium, reprogrammed CD34+ cells should be fed every-other-day with StemFlex Medium.
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We have evaluated the use of Cytotune-iPS 2.0 Sendai Reprogramming Kit (Cat. No. A16517) for somatic cell reprogramming of both neonatal and adult human dermal fibroblasts. For fibroblasts, follow the instructions provided in the Cytotune 2.0 reprogramming manual for feeder-free reprogramming (pgs. 16-20). On Day 7, you may use rhVTN-N (Cat. No. A14700), Geltrex matrix (Cat. No. A1413302), or rhLaminin-521 (Cat. No. A29248 or A29249). From Day 8 onward, rather than feeding daily with Essential 8 Medium, we recommend that you feed reprogrammed fibroblasts every-other-day with StemFlex Medium.
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We have not yet evaluated the Geltrex matrix system for clonal expansion in the presence of StemFlex Medium. However, rhLaminin-521 does provide optimal survival of cells following single-cell passaging and thus this matrix is recommended for such critical applications.
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We recommend use of the Neon Electroporation device for electroporation of PSCs with Cas9 protein:guide RNA complex following the protocol guidance in the following demonstrated protocol (https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/PG1584-PJT1313-COL31227-Demonstrated-Protocol-Performing-CRISPR-Cas9-FHR.pdf); see the section entitled “Knockout by electroporation of RNP using the Neon Transfection System.” We have seen that the Neon electroporation protocols 7 and 14 provide optimal indel formation while maintaining cell survival. However, the electroporation conditions may need to be optimized for your pluripotent cell line.
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Yes. We recommend following the coating instructions for Vitronectin or rhLaminin-521 and using the culture and passaging recommendations in the StemFlex protocol.
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The use of ROCK inhibitor is not required when culturing in StemFlex Medium on rhLaminin-521. However, supplementation with RevitaCell Supplement can provide additional support to PSCs during stressful transitions such as single-cell passaging.
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Yes. PSCs cryopreserved from cultures of mTeSR Medium and BD Matrigel Basement Membrane Matrix may be thawed into Gibco Essential 8 Medium and plated on VTN-N. Certain lines may benefit from thawing into the medium and substrate they were growing in at the time of cryopreservation. Then at the next passage, use EDTA to passage the cells into Gibco Essential 8 Medium and VTN-N.
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There have been multiple pluripotent stem cell (PSC) lines tested with the Gibco Essential 8 Medium System (https://www.ncbi.nlm.nih.gov/pubmed/?term=21478862).
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bFGF is vital for pluripotent cell survival and proliferation.
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We have tested and confirmed utility with the following PSC growth media: Gibco Essential 8 Medium, Gibco StemPro hESC SFM, mTesR1 medium, and Gibco KnockOut Serum Replacement (KSR)- containing feeder-dependent medium.
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Yes, fibroblasts from skin biopsy samples can be expanded and cultured in Gibco Essential 8 Medium with the addition of EGF, thrombin, and hydrocortisone (http://www.ncbi.nlm.nih.gov/pubmed/21478862).
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Insulin is important for cell survival and proliferation.
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Yes, cells can be routinely frozen in complete Gibco Essential 8 Medium and 10% DMSO. Alternatively, ready-to-use CTS PSC Cryomedium (Cat. No. A4238801) provides more efficient post-thaw recovery.
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Enzymes such as dispase and collagenase do not work well with cells cultured in Gibco Essential 8 Medium on VTN-N. Use of these enzymes for passaging cells results in compromised viability and attachment.
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Cells cultured in Gibco Essential 8 Medium and VTN-N need to be passaged with EDTA.
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No. The cells should be fed daily including the day after passaging.
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Yes, ROCK inhibitors can be used with Essential 8 Medium. We suggest using RevitaCell Supplement (Cat. No. A2644501), which has been specifically designed to minimize the impact of stress on PSCs.
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It is very important that complete Gibco Essential 8 Medium is prewarmed at room temperature and not in a 37 degrees C water bath. bFGF activity can decline rapidly with repeated temperature changes from 4 degrees C to 37 degrees C.
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The shelf life of complete Gibco Essential 8 Medium is two weeks at 2-8 degrees C.
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We recommend thawing the supplement at room temperature for approximately 1 hour.
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Other catalog versions of DMEM/F-12 cannot be used in place of the Gibco Essential 8 Basal Medium in the preparation of Gibco Essential 8 Medium. Gibco Essential 8 Basal Medium supplied with the kit has a higher level of sodium bicarbonate.
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To prepare 500 mL of complete Gibco Essential 8 Medium, thaw Gibco Essential 8 Supplement (50X) at room temperature for 1 hour and then aseptically combine the components listed below:
- Gibco Essential 8 Basal Medium: 490 mL
- Gibco Essential 8 Supplement (50X): 10 mL
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You should expect to see normal pluripotent stem cell (PSC) morphology. The expected morphology of PSCs is demonstrated specifically by tightly packed colonies with defined borders and a high nucleus-to-cytoplasm ratio.
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Cells cultured in other feeder-free media systems, such as mTeSR Medium with Matrigel Basement Membrane Matrix, or StemPro hESC SFM with Geltrex Matrix, can be successfully cultured in Gibco Essential 8 Medium and VTN-N. In addition, PSCs grown on feeders with KnockOut SR have also been shown to be successfully cultured in Gibco Essential 8 Medium on VTN-N. However, when changing media systems, cells must be passaged either manually, or with EDTA prior to culturing in Gibco Essential 8 Medium on VTN-N.
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Gibco Essential 8 Medium has reduced variability compared to existing feeder-free culture media. Unlike other media that contain over 20 highly variable ingredients, Gibco Essential 8 Medium is produced under cGMP and has an optimized formulation and growth factor levels that help ensure maximum cell health, pluripotency, and growth, with minimal variability.
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Gibco Essential 8 Medium and vitronectin have been shown to support PSC growth for >50 passages without any signs of karyotypic abnormalities, and maintain the ability of PSCs to differentiate into all three germ line lineages. As published by Chen et al (http://www.ncbi.nlm.nih.gov/pubmed/21478862) in the laboratory of James Thomson, the VTN-N variant of vitronectin supports human pluripotent stem cell attachment and survival better than wild-type vitronectin when used in conjunction with Gibco Essential 8 Medium.
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Yes. Gibco Essential 8 Medium provides reliable and robust cultures with a xeno-free, eight-component medium
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Here are three major differences to be taken into consideration when culturing cells in Gibco Essential 8 Medium on Gibco Vitronectin (VTN-N) compared to other feeder-free systems:
- Cells should be typically passaged ~24 hours sooner than they would be with other feeder-free media.
- Passaging should take place when cells are at ~85% confluency. If cells are passaged when they are more than 85% confluent, the health of the cells and final cell yield may be compromised.
- Cells must be passaged in EDTA. Collagenase and dispase are not recommended.
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Yes. Gibco Essential 8 Medium contains 100 ng/mL basic fibroblast growth factor (bFGF), and no additional bFGF is required.
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Gibco Essential 8 Medium contains only the eight required components for culturing PSCs. The medium was developed by Chen et al (http://www.ncbi.nlm.nih.gov/pubmed/21478862) to overcome the variability issues observed with mTeSR Medium. Gibco Essential 8 Medium is designed to have less variability due to limited components and removal of albumin (BSA) from the formulation. Gibco Essential 8 Medium is provided as a convenient two-component kit: 500 mL Gibco Essential 8 Basal Medium and 10 mL Gibco Essential 8 Supplement (50X).
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Gibco Essential 8 Medium is a xeno-free and feeder-free medium specially formulated for the growth and expansion of human pluripotent stem cells (PSCs). Originally developed by Chen et al (http://www.ncbi.nlm.nih.gov/pubmed/21478862) in the laboratory of James Thomson, and validated by Cellular Dynamics International, Gibco Essential 8 Medium has been extensively tested and is proven to maintain pluripotency in multiple PSC lines.
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Yes, you may use EDTA or versene. However, we do not recommend using dispase or collagenase as it can lead to differentiation.
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We recommend that you leave the product on ice while prepping to use it.
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Yes, do not let the laminin dry out on the plates.
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Yes. Following 2 passages on the rhLaminin-521 matrix, Versene or EDTA passaging should be used to subculture PSCs.
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The optimal working concentration of rhLaminin-521 is cell line dependent and must be determined empirically. However, for some cell lines, coating concentrations as low as 0.3 µg/cm2 can be used with no decrease in performance. Additionally, coating plates overnight at 4 degrees C can support coating concentrations as low as 0.1 µg/cm2.
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Yes. To ensure optimum recovery of PSCs following single-cell passaging, PSCs should be fed with Essential 8 Flex Medium the day before passaging.
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