![]() ![]() E8/PVA is used for EB formation ( Ng et al., 2008). PVA containing media: Polyvinyl Alcohol (PVA) powder is added into specific medium (E8 or E6) at 4 mg/ml, mixed and stirred for one hour at room temperature, filtered and then stored at 4☌. as Essential 6, or from Stem Cell Technologies as TeSR-E6. Similar medium is available from Thermal Fisher/Life Technologies Inc. The medium could be prepared based on the formula in previous publication ( Chen et al., 2011). In this protocol, we simplify all such hPSC maintenance media as E8.Į6/Essential 6 medium: E6 is similar to E8 medium but without FGF2 and TGF-β. as Essential 8, or from Stem Cell Technologies as TeSR-E8. As an alternative, the medium is also available from Thermal Fisher/Life Technologies Inc. Vitronectin coated plate/dish: Apply the same procedure of Matrigel coating to coat vitronectin, 2 mg vitronectin is used to coat 10 six-well plates or 10 10 cm dishes.Į8/Essential 8 media: The medium could be prepared based on the formula in previous publication ( Chen et al., 2011). The Matrigel-coated plates/dishes can be sealed and stored at 4☌ for more than one week before use. Leave the plates/dishes at room temperature for at least 30 minutes or at 4☌ overnight. 4 mg Matrigel is used to coat 5 six-well plates or 5 10 cm dishes. Mix the Matrigel with the media well, plate 6 ml in one six-well plate (1 ml per well) or 6 ml in one 10 cm dish and shake well to cover the entire surface. Matrigel coated plate/dish: Pour 30 ml cold DMEM/F12 in conical tube, use 1.5 ml to resuspend 4 mg frozen Matrigel with 5 ml pipet. In this protocol, we describe a set of optimized procedures to produce EBs from human PSCs in E8 or E8-based media.ĮDTA/PBS solution: dilute 0.5 ml 0.5M EDTA (pH 8.0) in 500 ml PBS (calcium and magnesium free), add 0.9g NaCl, mix and filtrate. We previously developed a fully chemically defined medium Essential 8 (or E8) for the maintenance and expansion of human pluripotent stem cells in the clinical grade environment. This will allow better consistency as well as an easier route to translate into clinically relevant production. As such, it is essential that EB formation and further differentiation can be conducted in chemically defined, animal product-free conditions. Meanwhile, unlike their mouse counterparts, human PSCs usually cannot survive in suspension unless in aggregates or under ROCK inhibitor treatment. This inconsistency also severely affects researchers’ ability to further improve procedure and its final products. At the same time, the undefined composition of the above components can lead to inconsistent outcomes in experiments due to batch differences in their production. These animal-sourced components significantly limit the application of EB formation to generate potentially clinically relevant cell products. However, most EB formation protocols contain undefined components, such as Fetal Bovine Serum (FBS), Knock-Out Serum Replacement (KOSR) or albumin product. EB differentiation is a common platform to generate specific cell lineages from PSCs. Embryoid bodies (EB) are the three-dimensional aggregates formed in suspension by pluripotent stem cells (PSC), including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC). ![]()
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