![]() ![]() The methods are specific for the species, lineage, and cell type. Therefore, many protocols have been developed for directly differentiating ESCs and iPSCs without using EBs. When a specific lineage or cell type is required, cultures must be depleted of the unwanted cell types. Miguel Esteban.ĭepending on the desired end point, the presence of all three germ layers in EBs can be a disadvantage. All three germ layers are present, but the composition of the media influences the ratios of cell types and lineages.Įmbryoid bodies after growing in suspension for eight days. This method is efficient for making EBs, but is not as scalable as suspension culture.ĮSCs form EBs in about four days, and are often allowed to grow for weeks. Semisolid media, usually methylcellulose, can be used to suspend the EBs. Now, higher-throughput hanging-drop methods using arrays have been developed and used for anticancer-drug sensitivity testing (Hsaio et al. Making the drops (usually ~20 μl) was formally labor intensive, and hence the numbers of EBs generated was low. The size of the EBs can be controlled by controlling the number of cells in each drop. Hanging-drop culture gives smaller and much more uniform EBs. Suspension culture is scalable to bioreactors, although the exact methods and factors such as stir rate that affect the physical dimensions of EBs must be determined empirically. Suspension culture is the most common method for EB formation but also the hardest to control, for both EB size and shape EBs can become large and irregularly shaped in suspension. All protocols start with detaching a high-density cell culture from the dish, either enzymatically or with versine, depending on the properties of the cells. There are several methods for forming EBs, including suspension culture, hanging-drop culture, and culture in semisolid media. All downstream differentiated cells are derived from this initial structure. An EB contains all three germ layers.ĮBs are still often used as the initial stage of differentiation for embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). These aggregated cells spontaneously differentiate. Generally, when stem cells are cultured without an adherent surface, feeder cells, or a complex matrix, the cells aggregate. One of the oldest methods for stem cell differentiation is the generation of embryoid bodies (EBs).
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