Development of microcapsules for producing spherical tissues of < 150 μ m in size

Cell-enclosing spherical microcapsules have been studied as a basic research tool, therapeutic device for tissue transplantation, and production of useful proteins (Uludag 2000). We have previously developed a method for preparing cell-enclosing microcapsules with solid core of less than 100 μm in diameter (Sakai 2004). The microcapsules are much smaller than conventional cellenclosing microcapsules of 500-1,000 μm in diameter. We prepared such small capsules via a droplets breakup technology in a laminar flow of water-immiscible liquid. In addition, we developed a technique producing cell-enclosing microcapsules with hollow core of ca.100-150 μm in diameter using the small microcapsules with solid core as template of the liquid cores (Sakai 2008). The size of liquid cores was less than the maximum allowable capsule size of 260 μm in diameter for a tightly packed cell mass that does not limit the oxygen supply resulting in necrotic region formation inside spherical tissues (Kim 1998). Previously (Sakai 2008), we enclosed mouse embryonic stem cells in the microcapsules with 20-50 μm of agarose gel membrane and with hollow core of 100-150 μm in diameter. The cells grew and formed embryoid body-like spherical tissues having the same size with the hollow cores (Sakai 2008). In some applications such as tissue engineering, it is required to collect the spherical tissues from microcapsules without damaging them. However, in this point, agarose gel is not a suitable material for microcapsule membrane. For removing agarose gel membrane, we have to heat them more than 60°C. In addition, in general, agarase, an enzyme of digesting agarose molecules, can not liquefy agarose gel under the condition mild for mammalian cells. In this paper, we attempted to develop microcapsules with hollow cores of < 150 μm in diameter and degradable membrane under mild condition for mammalian cells for collecting spherical tissues formed in the vehicles.