Ichiro Hashimoto

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 microm in diameter were developed to obtain multicellular spheroids with well-defined sizes of less than 150 microm 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 H(2)O(2), resulting in cell-enclosing CMC-Ph microparticles, 135 microm 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 Sr(2+). 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.

Subsieve-size agarose capsules enclosing ifosfamide-activating cells: a strategy toward chemotherapeutic targeting to tumors

Localized activation of the prodrug ifosfamide in or close to tumors by implanting encapsulated ifosfamide-activating cells is an efficacious strategy for tumor therapy. The aim of this study was to evaluate the feasibility of subsieve-size agarose capsules for enclosing the cells in this application. Compared with many conventional microcapsules, subsieve-size agarose capsules are about one-tenth the size and have both higher mechanical stability and allow better molecular exchangeability than other systems. Cells that have been genetically modified to express cytochrome P450 2B1 enzyme were encapsulated in subsieve-size agarose capsules of ∼90 μm in diameter and implanted into preformed tumors in nude mice. Living cells were detected for >1 month after encapsulation in vitro and showed enzymatic activity (i.e., they were able to activate ifosfamide). More significant regression of preformed tumors was observed in the recipients implanted with cell-enclosing capsules compared with those implanted with empty capsules. These results suggest that the strategy of using subsieve-size agarose capsules enclosing cytochrome P450 2B1–expressing cells is feasible for tumor therapy by chemotherapeutic targeting in combination with ifosfamide administration.

Small agarose microcapsules with cell-enclosing hollow core for cell therapy: transplantation of Ifosfamide-activating cells to the mice with preestablished subcutaneous tumor.

Cell transplantation after enclosing in microcapsules has been studied as an alternative approach for treatment of wide variety of diseases. In the present study, we examined the feasibility of using agarose microcapsules, having a cell-enclosing hollow core of 100–150 μm in diameter and agarose gel membrane of about 20 μm in thickness, as a device for the methodology. We enclosed cells that had been genetically engineered to express cytochrome P450 2B1, an enzyme that activates the anticancer prodrug ifosfamide. The enclosed cells were shown to express the enzymatic function in the microcapsules in that they suppressed the growth of tumor cells in medium containing ifosfamide. In addition, a more significant regression of preformed tumors was observed in the nude mice implanted with the cell-enclosing microcapsules compared with those implanted with empty capsules after administration of ifosfamide. Preformed tumors shrank by less than 40% in volume in 6 of the 10 recipients implanted with cell-enclosing microcapsules. In contrast, only 1 in 10 of the preformed tumors in the recipient implanted with empty microcapsules shrank by this amount. These results suggest that agarose microcapsules containing cytochrome P450 2B1 enzyme-expressing cells are feasible devices for improving the chemotherapy of tumors. Thus, agarose microcapsule having hollow cores are generally a good candidate as vehicles for cell-encapsulation approaches to cell therapy.

Agarose-gelatin conjugate membrane enhances proliferation of adherent cells enclosed in hollow-core microcapsules

Controlling growth of cells enclosed in hollow-core microcapsules is an important issue for the practical use of the device in biomedical and biopharmaceutical fields. In this study, we developed hollow-core microcapsules with a cell-adhesive agarose–gelatin conjugate (Aga–Ge) gel membrane for enhancement of adherent cell growth. We enclosed adherent feline kidney cells in these microcapsules and compared their growth profile and behavior with cells in microcapsules with an unmodified agarose membrane. The cells grew approx. 2-fold faster in microcapsules with the Aga–Ge membrane than in those with the unmodified agarose membrane. Fluorescence observation of the cellular skeleton clearly revealed that the enclosed cells adhered and spread on the inner surface of the Aga–Ge membrane but not on the unmodified agarose membrane. The maximum cell densities estimated on the basis of the cellular mitochondrial activities were independent of the cellular adhesiveness of the membrane. The mitochondrial activities per vehicle were similar for the two types of microcapsules. These results demonstrate that construction of microcapsule membranes from cell-adhesive materials is effective for enhancing cellular growth in these devices.