Embryonic stem cell (ESC) culture is fragmented, laborious and involves operator decisions. Most protocols consist of three individual steps: a) maintenance, b) embryoid body (EB) formation and c) differentiation. Achieving integration will assist automation, which can ultimately result in scale-up to clinically relevant numbers. Bioreactors provide a dynamic cultivation system within a controlled environment that enables the expansion of cell populations. Suspension culture systems offer attractive advantages of scalability and relative simplicity that can improve the viability and turnover of specific stages and types of stem cells (1). However, 2D culture, in which normal 3D relationships with the extracellular matrix and other cells are distorted, may alter cellular behaviour. Analysing cell interactions in more natural 3D settings promises to provide a view that is closer to what actually occurs in vivo (2,3). Recently, the use of bioreactors for human ESC culture has been documented and offered evidence that dynamic, 3D conditions provide an advantageous environment for the culture of ESCs (4). Nonetheless, to our knowledge, no current methodologies exist that allow the integrated maintenance, expansion and differentiation of ESCs towards the osteogenic lineage forming 3D mineralised tissue. Alginate encapsulation has been carried out frequently with adult cells and more recently, Magyar et al. (5) encapsulated mESCs that were able to form discoid colonies, cystic EBs, and smooth muscle cells. Chondrogenic differentiation from mesenchymal stem cells encapsulated in alginate beads has been reported (6) and a combination of alginate with gelatin has been considered to provide a biodegradeable delivery vehicle for tissue engineering applications (7). Normally, alginate hydrogels lose Ca²⁺ cations after prolonged culture, but the incorporation of gelatin enables cell-mediated contraction and packing of the scaffold material (8). The use of alginate to enhance chondrogenesis from encapsulated EBs derived from mESCs has been attempted, albeit with limited success (9). In this work, we present a simplified, integrated, and reproducible bioprocess for the production of osteogenic cells from mESCs that would be amenable to automation and scale-up for the generation of clinically relevant numbers of high-quality bone cells and mineralised tissue. Specifically, we encapsulated mESCs in alginate hydrogels, cultured them in horizontal aspect ratio vessels (HARV) and adapted our previously established protocols (10) to induce osteogenic differentiation. In this one-step, integrated process, we differentiated mESCs into osteogenic cells capable of producing 3D mineralised tissue identified by demonstration of stained mineralised aggregates, the expression of markers such as osteocalcin, OB-cadherin and collagen type-I, and micro-CT.

References

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