A precise spatial multicellular polymer hydrogel matrix was prepared by successive assembly of cell-laden hydrogel layers alternated by hydrogel layers without cells based on the spontaneous hydrogel formation between 2 aqueous polymer solutions. The polymers used were a water-soluble 2-methacryloyloxyethyl phosphorylcholine polymer bearing phenylboronic acid groups (PMBV) and poly(vinyl alcohol) (PVA). Each cell-laden layer was deposited as a cell-laden PMBV solution on a PMBV/PVA precursor film. PMBV/PVA multilayer hydrogel was stacked on the top of a cell-laden layer by sequential coating with spinning of the PMBV and PVA solutions. This process allowed the formation of the PMBV/PVA multilayer hydrogel with finely controlled thickness. Finally, we succeeded in cell patterning by using a multilayer hydrogel matrix, forming a sandwich of 2 cell-laden layers separated by a PMBV/PVA multilayer hydrogel. The cells remained alive during the spinning process and maintained their metabolism for at least 24. h. This precise spatial multicellular PMBV/PVA hydrogel can be used to examine interactions between many different cells and construct customized microenvironments for multicellular co-cultures.
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