Organ manufacturing is a promising method to replace failing complex organs. In this study, a new cell cryopreservation technique was developed with cryoprotectants being directly incorporated into the cell/hydrogel construct according to predesigned structures and then subjected to a special freezing/thaw process. The hydration of the gelatin/alginate hydrogel was greatly increased while the eutectic temperature was decreased by the adding glycerol. The addition of dextran-40 was found to also effectively improve the cell survival when incorporated with glycerol. The most favorable cryoprotectant concentration was 2.5% glycerol with 5% dextran-40 in a gelatin/alginate hydrogel. Under these conditions, the cell viability in the construct was >95%. Microscopic observations, MTT analysis, hematoxylin and eosin staining indicated that the cells proliferated immediately after thawing. The cells in the gelatin/alginate hydrogel with both glycerol and dextran-40 had proliferated more than with only glycerol or dextran-40 alone. This approach can be adapted for use in cell assembly in high-throughput drug screening and complex organ manufacturing areas.
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