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By preventing mesenchymal stem cells (MSCs) from adhering to precoated agarose to create a model of MSC suspension in vitro, we investigated anoikis in MSCs and the role of caspase-3 in the anoikis. The cultured MSCs were randomly divided into 3 groups: the anoikis group,caspase-3 inhibitor group and control group. Before experiment, we coated dishes with 1.5 % agarose;in the anoikis group, MSCs were put into the precoated dishes; and in the inhibitor group, caspase-3 inhibitor and MSCs were also put into the precoated dishes; but there were not intervention in the control group. MSCs were collected at 2 h, 6 h, 12 h and 24 h. The alteration of caspase-3 activity was evaluated by caspase-3 fluorometric assay and west blot analysis. The apoptosis rates were detected by flow cytometry. MSCs were round and suspended sufficiently in the anoikis and inhibitor groups. Caspase-3 fluorometric assay showed that there were significant differences in statistics between the anoikis group and the others (P＜0.05). West blot analysis discovered that caspase-3 expression.in the anoikis group was more than that in the control and inhibitor groups (P＜0.05). Flow cytometry showed that the apoptosis peak appeared in all the three groups, but it increased dramatically in the anoikis group. The apoptosis rates in the inhibitor and control groups were low and stable.And there were significant differences in statistics between the anoikis group and the others (P＜0.05).MSCs will undergo anoikis in suspended condition if they are separated from the extracellular matrix.Caspase-3 inhibitors can suppress caspase-3 activity and reduce the apoptosis rate significantly. Caspase-3 plays a vital part in induced MSC anoikis in vitro. MSCs suspension culture system might be set up with argorose and caspase-3 inhibitor.