Spheroid-based cell delivery has been demonstrated as an effective strategy in stem cell therapy of critical limb ischemia. However, massive generation of uniform-sized spheroids still remains a big challenge. In addition, how spheroid size influences its therapeutic efficacy remains an open question. Herein, patterned poly(2-hydroxyethyl methacrylate) hydrogel films synthesized in situ in cell culture plates are used to generate adipose-derived stromal cell (ADSC) spheroids. This method not only allows for the production of uniform-sized spheroids but also large-scale production (≈60 000 spheroids per plate). Using this method, monodisperse ADSC spheroids with three different sizes are generated to study spheroid size effect in ischemia treatment. It is found that different size spheroids exhibit different behaviors in extracellular matrix (ECM) expression and paracrine secretion. Larger spheroids express more ECM proteins. Paracrine secretion of hypoxia-induced survival factors, anti-apoptotic and angiogenic factors is enhanced to a larger degree in larger spheroids. Finally, using a mouse model of hind limb ischemia, it is demonstrated that the therapeutic efficacy of the spheroids increases with increasing spheroid size. The improved therapeutic efficacy is attributed to enhanced survival and paracrine secretion of larger spheroids, which is attributed to the higher degree of hypoxic environment of these spheroids.
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