Multicellular spheroids (MCSs) are in vitro tissue models having important biomedical applications. The MCS generation is usually time-consuming because most MCS fabrication methods exploit the inherent ability of cells to self-assemble to form spheroids. Here we tried to accelerate the MCS generation by chemical pre-aggregation of the cells. We first developed a method to chemically aggregate cells, which involves a first treatment of cells with NaIO4 to produce surface aldehyde functionalities, and then incubation with acrylic acid-modified chitosan (chitosan-AA), a new inter-cellular linker. Biocompatibility test indicates chitosan-AA is non-toxic to cells, even at a high concentration. When incubated in the presence of chitosan-AA, the NaIO4-treated cells aggregate quickly, because of the reaction between the amino groups on chitosan-AA and the aldehyde groups on cell surface. In addition, the cells in the resulting aggregates remain a high viability. The pre-aggregated cells were then seeded into an in situ-formed hydrogel to generate MCSs. While it takes several days to form MCSs in the control groups, it takes only 1 day for the cells to form compact spheroids in the pre-aggregation group. The spheroids formed in the pre-aggregation group (231 +/- 74 mu m at Day 4) are bigger than those obtained in the control groups (70 +/- 17 mu m at Day 4). In addition, the cells in the pre-aggregation group grow and proliferate faster (P < 0.05), because of the improved cell-cell interaction in the aggregates. (C) 2015 Elsevier Ltd. All rights reserved.