Abstract
BackgroundA fundamental requirement for the exploitation of embryonic stem (ES) cells in regenerative medicine is the ability to reproducibly derive sufficient numbers of cells of a consistent quality in a cost-effective manner. However, undifferentiated ES cells are not ideally suited to suspension culture due to the formation of cellular aggregates, ultimately limiting scalability. Significant advances have been made in recent years in the culture of ES cells, including automated adherent culture and suspension microcarrier or embryoid body bioreactor culture. However, each of these methods exhibits specific disadvantages, such as high cost, additional downstream processes or reduced cell doubling times.Methodology/Principal FindingsHere we show that abrogation of the cell surface protein E-cadherin, using either gene knockout (Ecad-/-) or the neutralising antibody DECMA-1 (EcadAb), allows culture of mouse ES cells as a near-single cell suspension in scalable shake flask culture over prolonged periods without additional media supplements. Both Ecad-/- and EcadAb ES cells exhibited adaptation phases in suspension culture, with optimal doubling times of 7.3 h±0.9 and 15.6 h±4.7 respectively and mean-fold increase in viable cell number of 95.1±2.0 and 16±0.9-fold over 48 h. EcadAb ES cells propagated as a dispersed cell suspension for 15 d maintained expression of pluripotent markers, exhibited a normal karyotype and high viability. Subsequent differentiation of EcadAb ES cells resulted in expression of transcripts and proteins associated with the three primary germ layers.Conclusions/SignificanceThis is the first demonstration of the culture of pluripotent ES cells as a near-single cell suspension in a manual fed-batch shake flask bioreactor and represents a significant improvement on current ES cell culture techniques. Whilst this proof-of-principle method would be useful for the culture of human ES and iPS cells, further steps are necessary to increase cell viability of hES cells in suspension.
Highlights
Embryonic stem (ES) cells, with their self-renewal ability and multiple lineage differentiation capacity, are attractive for many applications in regenerative medicine and drug screening
Static Suspension Culture of Wt and E-cad-/- embryonic stem (ES) Cells We have previously shown that Ecad-/- ES cells exhibit loss of cell-cell contact and are able to self-renew in serum-supplemented medium in the absence of leukaemia inhibitory factor (LIF) [17]
Ecad-/- ES cell cultured in static suspension culture exhibited weak cell-cell interaction and failed to establish characteristic embryoid body (EB) morphology compared to wtEBs (Figure 1A(i) and (ii), respectively)
Summary
Embryonic stem (ES) cells, with their self-renewal ability and multiple lineage differentiation capacity, are attractive for many applications in regenerative medicine and drug screening. An alternative method is the embryoid body (EB) cultivation method, which utilises shear stress to control aggregate size [7,14,15] and may contain enzymatic dissociation steps to prolong culture times [16] This approach is disadvantaged by diffusion limitations within individual EBs leading to EB agglomeration and less efficient cellular expansion compared to conventional culture methods. Significant advances have been made in recent years in the culture of ES cells, including automated adherent culture and suspension microcarrier or embryoid body bioreactor culture Each of these methods exhibits specific disadvantages, such as high cost, additional downstream processes or reduced cell doubling times
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