Gel-matrix culture environments provide tissue engineering scaffolds and cues that guide cell differentiation. For many cellular therapy applications such as for the production of islet-like clusters to treat Type I diabetes, the need for large-scale production can be anticipated. The throughput of the commonly used nozzle-based devices for cell encapsulation is limited by the rate of droplet formation to similar to 0.5 L/h. This work describes a novel process for larger-scale batch immobilization of mammalian cells in alginate-filled hollow fiber bioreactors (AHFBRs). A methodology was developed whereby (1) alginate obstruction of the intracapillary space medium flow was negligible, (2) extra-capillary alginate getting was complete and (3) 83 +/- 4% of the cells seeded and immobilized were recovered from the bioreactor. Chinese hamster ovary (CHO) cells were used as a model aggregate-forming cell line that grew from mostly single cells to pancreatic islet-sized spheroids in 8 days of AHFBR culture. CHO cell growth and metabolic rates in the AHFBR were comparable to small-scale alginate slab controls. Then, the process was applied successfully to the culture of primary neonatal pancreatic porcine cells, without significant differences in cell viability compared with slab controls. As expected, alginate-immobilized culture in the AHFBR increased the insulin content of these cells compared with suspension culture. The AHFBR process could be refined by adding matrix components or adapted to other reversible gets and cell types, providing a practical means for gel-matrix assisted cultures for cellular therapy. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 1740-1751, 2009