Cuanticación del mecanismo de apertura de la doble hebra

For the purposes of functional tissue replacement, it has been estimated that upwards of 10⁹ cells will be necessary for regenerative therapies (Tzanakakis et al. 2000, Lock and Tzanakakis 2007, Jing et al.

2008). Therefore, the development of methods for increasing the scale of ESC culture is important to produce clinically relevant yields. In their pluripotent state, ESCs are typically cultured in monolayer, which restricts the number of cells produced, dependent on the culture surface area. Some groups have established methods for using beads made of alginate or other adhesive materials, to increase the sur-face area for growth (Fernandes et al. 2007, Lock and Tzanakakis 2009). Alternatively, when cultured as small aggregates in the presence of LIF or fibroblast conditioned media, ESCs maintain pluripotency (Cormier et al. 2006, Ouyang et al. 2007).

Suspension culture is amenable to scale-up for larger volume cultures, such as bioreactors (Gerecht-Nir et al. 2004, Bauwens et al. 2005, Fok and Zandstra 2005, Cameron et al. 2006, Wang et al. 2006, Zur Nieden et al. 2007, Niebruegge et al. 2008, Krawetz et al. 2010). Common formats include spinner flasks, rotating wall vessels (high aspect rotating vessel, slow turning lateral vessel) and large-scale bioreactors. Large-volume culture systems are also amenable to modifications for increased monitor-ing and control of the culture environment, which is important for uniformity between culture envi-ronments (Côme et al. 2008, Gerlach et al. 2010). Large-scale culture systems, however, all necessitate mixing to prevent gradients of nutrients and waste within the culture (Bilgen et al. 2005, 2006, Sargent et al. 2010). Hydrodynamic mixing within cultures may impact EB homogeneity, viability, and differ-entiation (Carpenedo et al. 2007, Sargent et al. 2010). Therefore, it is important to consider the impact of the environmental changes necessary for increasing the scale of cultures for clinical settings.

8.7 Conclusion

Although hESCs are a promising cell source for many translational applications, much remains to be determined regarding the proteomic, genetic, and epigenetic mechanisms that regulate pluripotency and differentiation. ESCs, therefore, may provide a useful platform to understand embryonic devel-opmental events that cannot be perturbed in vivo due to lethality of genetic knockouts. For example, cell lines transduced with reporter constructs allow real-time monitoring of genetic changes during differentiation and are amenable to high-throughput screening approaches. iPS may, additionally, provide insight into regulatory mechanisms, specifically with respect to the epigenetic changes dur-ing reprogrammdur-ing of somatic cells. The derivation of individualized iPS lines provides methods for understanding the pathophysiology of disease-specific cell lines, and as platforms for drug test-ing and autologous transplantation. From an engineertest-ing perspective, understandtest-ing the interac-tions of ESCs with their environment will enable the development of modified culture approaches to modulate cell potential through cell–cell adhesions, morphogen delivery, and mechanical fac-tors. Understanding the ESC microenvironment may also enable alternative therapeutic approaches, including harnessing the paracrine potential of secreted growth factors and ECM for directing somatic cell functions in vivo.

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In document Estudio a nivel de moléculas individuales de la actividades de la actividad de replicación del ADN de la polimerasa del bacteriófago Phi29 (página 66-74)