Protocol 9 continued

Medium analysis:

8 Remove triplicate 1 ml samples from the conditioned medium, and immediately analyse for pO2, pCO2, pH, glucose, and lactic acid levels using a Blood Gas Analyzer (StatProfile M, Nova Biomedical); or store at -70 °C for subsequent analysis of lactate dehydrogenase (LDH) activity using a Chemistry Analyzer (Roche Hitachi Modular P800).

a At this speed the suspended cell aggregates remain close to a stationary point within the bioreactor vessel.

enhancement in their yield compared to EBs produced by static methods. Dynamically formed EBs exhibit steady and progressive differentiation, with cyst formation and elaboration of complex structures such as neuroepithelial tubes, blood vessels, and glands (13; Figure 9B-D) as observed in statically formed EBs. Overall, we consider this to be the system of choice for controlling the aggregation and EB-mediated differentiation of hES cells, and for producing relatively large numbers of EBs in a reproducible manner, with minimal handling involved.

6.2 Production of hES cell-derived EBs within three-dimentional matrices

Another approach to guiding the differentiation of hES cells of relevance to scale-up procedures involves manipulating EBs by imposing physical constraints on their formation and growth; cells in EBs are responsive to physical, in addition to chemical, cues. Such confinement may be achieved by the encapsulation of hES cells in agarose hydrogel capsules (46), which permits culture at high cell density, enables EB formation, and allows differentiation into haematopoietic cell lineages. Recently, this system was successfully applied to enhance cardiomyocyte derivation from mES cells under hypoxic conditions (53).

Another confining environment is the three-dimensional porous scaffold; culture of hES cells within an alginate scaffold of this kind enables efficient formation of EBs with a relatively high degree of cell proliferation and differentiation (54). Under these conditions, EBs form mainly within the scaffold pores, and become distributed evenly over the entire scaffold volume. Most probably, the relatively small pore size together with the hydrophilicity of the alginate scaffold facilitate the generation and dispersal of EBs in this way. Scanning electron micrographs of the hES cell-seeded scaffolds recorded after 1 month of culture reveal that the forming EBs occupy the entire pore volume (Figure 10A), whilst some cells form a lining along the scaffold fibres (Figure 10B). Furthermore, this confining environment induces vasculogenesis in the differentiating EBs to a higher degree than observed in either static, suspension cultures, or in dynamic, rotating cultures (54).

Figure 10 EB formation within three-dimensional scaffolds. Scanning electron micrographs of LF120 alginate scaffolds seeded with hES cells and cultured for 1 month: (A) EBs (dashed arrows) develop mainly within pores confined by the scaffold fibres (solid arrows), whilst (B) other cells (dashed arrows) become disposed along the scaffold fibres (solid arrows).

Figure 10 EB formation within three-dimensional scaffolds. Scanning electron micrographs of LF120 alginate scaffolds seeded with hES cells and cultured for 1 month: (A) EBs (dashed arrows) develop mainly within pores confined by the scaffold fibres (solid arrows), whilst (B) other cells (dashed arrows) become disposed along the scaffold fibres (solid arrows).

Polymer scaffolds also have been shown to provide a conducive and supportive environment for the organization of early differentiating hES cells into tissuelike structures: cells dissociated from day 8 EBs, seeded on synthetic scaffolds and treated with appropriate cytokines subsequently undergo organization into complex three-dimensional structures resembling, and with features characteristic of, embryonic tissues (55, 56).

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