2014), promotes reliable growth of hiPS cells in a feeder-free and defined environment. 2014) and single-cell cloning (Feng et al. The Cellartis iPSC Single-Cell Cloning DEF-CS Culture Media Kit, recognized for its suitability for genome engineering (Valton et al. Monolayer culture systems and colony systems differ in the growth patterns of the hiPS cells they support the former supports a sheet of cells and the latter multiple colonies. This type of improved culture system should be feeder-free and defined, as the use of feeder cells results in a more complicated workflow, more labor, inaccurate quantification, and an increased risk of heterogeneous cultures.Īdditionally, the ideal system should sustain a feeder-free hiPS cell monolayer culture while going through the gene editing technique of choice, and then further ensure survival and proliferation once single cells are plated and expanded for mutant allele identification. ![]() A culture system that supports single-cell cloning and expansion of hiPS cells could overcome the current barrier of poor outcomes for single cells. Unfortunately, gene editing protocols often subject stem cells to harsh conditions that compromise their survival (e.g., electroporation), a problem that is compounded by the innate challenges of single-cell culture. Promoting survival and proliferation at the single-cell level is critical for expansion of any clonal colonies containing the mutation of interest from the gene editing process. This single-cell cloning bottleneck occurs because delicate hiPS cells typically grow in colony- and/or feeder-dependent conditions, and re-plating as single cells removes survival and growth signals, reducing viability. Currently, it is difficult to treat isolated clonal cells in a manner that allows them to survive the gene editing procedure and thrive. These methods are being applied in human-induced electroporation stem (hiPS) cells, for functional studies of genetic variation in disease models and for the larger field of regenerative medicine however, a major barrier remains at the level of selecting single cells with the desired mutations. Recent progress in gene editing technology has made it possible to introduce targeted genetic alterations in cells and organisms.
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