Despite the encouraging achievements of stem cell therapy in pre-clinical models, a recurring difficulty has been the inadequate retention and survival of the transplanted cells at target tissue, thus limiting the overall therapeutic benefit. Herein, through pre-assembly of cells into 3D hybrid spherical configuration by using methylcellulose hydrogel together with dextran and Ficoll that can induce macromolecular crowding (MMC), we are able to (i) maintain the cell–cell and cell–matrix interaction during the entire procedure of cell harvesting and transplantation and prevent anoikis, (ii) reduce cell leakage from the site of injection owing to the bulky volume of 3D hybrid cell spheroids, and (iii) endow engrafted cells against the harsh microenvironment presented in target tissue. Using a mouse model of ischemic stroke, we demonstrate that by employing the present platform, the critical underlying causes of cell loss during transplantation could be addressed effectively.

Comparing with conventional 2D monolayered culture, the 3D spheroid configuration is inherent with enhanced cell–cell and cell–ECM interactions that effectively recapitulate the microenvironment of native tissue, thus encouraging the secretion of soluble factors that contributes to therapeutic capability. Moreover, the enhanced ECM deposition induced by MMC can benefit the preservation of secreted soluble factors and have tremendous impact on multiple cellular behaviors that are crucial for post-engrafted cellular function and the resultant therapeutic efficacy. When transplanting stem cells in the configuration of 3D cell spheroids, the cells were administered together with the deposited ECM and soluble factors, thus promoting their subsequent survival.

Owing to the massive cell loss at various stages of cell transplantation, it required a large number of cells to be delivered for inducing significant therapeutic effect. By employing the presented platform that assembled cells into 3D hybrid spheroids, the retention, survival, and therapeutic potential can be enhanced tremendously, thus reducing the time and cost in preparing lots of cells for transplantation.