Enzymatically fabricated and degradable microcapsules for production of multicellular spheroids with well-defined diameters of less than 150 μm

Microcapsules with a single, spherical hollow core less than 150 μm in diameter were developed to obtain multicellular spheroids with well-defined sizes of less than 150 μm in diameter. An aqueous solution of phenolic hydroxyl derivative of carboxymethylcellulose (CMC-Ph) containing human hepatoma cell line (HepG2) cells and horse radish peroxidase (HRP) was injected into a coflowing stream of liquid paraffin, containing H₂O₂, resulting in cell-enclosing CMC-Ph microparticles, 135 μm in diameter, via a peroxidase-catalyzed crosslinking reaction. The CMC-Ph microparticles were then coated with a phenolic hydroxyl derivative of alginate (Alg-Ph) gel membrane several dozen micrometers in thickness, crosslinked via the same enzymatic reaction process, followed by further crosslinking between the carboxyl groups of alginate by Sr²⁺. A hollow core structure was achieved by immersing the resultant microcapsules in a medium containing cellulase, which degrades the enclosed CMC-Ph microparticles. The HepG2 cells in the microcapsules then grew and completely filled the hollow core. Multicellular spheroids the same size as the CMC-Ph microparticles, with living cells at their outer surface, were collected within 1 min by soaking them in a medium containing alginate lyase to degrade the Alg-Ph gel microcapsule membrane.