Preparation of Mammalian Cell-Enclosing Subsieve-Sized Capsules (<100 Mm) in a Coflowing Stream Shinji Sakai, Kenji Kawabata, Tsutomu Ono, Hiroyuki Ijima, Koei Kawakami Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581, Japan; telephone: +81-92-642-4109; fax: +81-92-642-4109; e-mail: sakai @chem-eng.kyushu-u.ac.jp Received 4 July 2003; accepted 4 November 2003 Published online 19 February 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20006 Abstract: The droplet breakup technique with an immis- cible liquid coflowing stream was investigated for the preparation of mammalian cell-enclosing subsieve-sized capsules of less than 100 Am in diameter. The major parts of the droplet generation device were a needle of several hundred micrometers in diameter for extruding the cell- suspending sodium alginate aqueous solution and a tubule of 2.5 mm in diameter through which the extruded alginate solution flowed into ambient immiscible liquid paraffin. The needle was positioned upstream in the vicinity of the coaxial tubule. The droplet diameter of the viscous sodium alginate aqueous solution could be controlled from several dozen to several hundred micrometers by changing the velocities of the inner and ambient fluids and the diameter of the needle. By utilizing a 300-Am diameter needle, CHO- K1 cell-enclosing droplets of 48 F 8 Am in diameter were obtained by extruding a cell-suspending sodium alginate solution at a velocity of 1.2 cm/sec into the ambient liquid paraffin flowing at a velocity of 23.5 cm/sec. The breakup process did not influence the viability of the enclosed cells, since more than 95% of the CHO-K1 cells remained alive after the enclosing process. B 2004 Wiley Periodicals, Inc. Keywords: coflowing stream; subsieve-sized capsule; cell therapy; microcapsule; alginate; laminar flow INTRODUCTION Recent advances in genetic engineering have enabled the use of cells as reactors to provide desired proteins. Implantation of recombinant cells after microencapsulation in semiper- meable membranes is one of the effective methods for in vivo delivery of proteins. Delivery of proteins has been investigated as a therapy for a wide range of diseases (Chang and Prakash, 1998; Chang et al., 1999; Prakash and Chang, 1996), such as tumors (Joki et al., 2001; Lohr et al., 2001; Read et al., 2001) and Huntington’s disease (Emerich et al., 1997). The basic principle of microencapsulation is the protection of the encapsulated cells from the host immune system. For this immunoprotection, the membrane has to exclude antibodies, complement proteins, some cytokines, and other cytotoxic substances. At the same time, it has to allow the permeation of substances such as oxygen, nutrients, and cellular metabolites necessary for the survival of the encapsulated cells. The therapeutic products of the encapsulated cells also have to be able to permeate through the membrane. The idea of using microcapsules for the immunoprotection of transplanted cells was proposed by Chang (1964). Lim and Sun (1980) substantiated its efficiency by implanting microencapsulated allogeneic rat islets. These microcapsules had diameters of several hundred mi- crometers to enclose the islets that were 50–300 Am in diameter microencapsulation has been progressed based on techniques developed for islet-enclosing bioartificial pancreas (Chang et al., 1999; Orive et al., 2003; Sakai et al., 2002; Uludag et al.,2000). Even for the encap- sulation of single cells of less than 30 Am in diameter, microcapsules with diameters in the range of several hun- dred micrometers to millimeters have been used (Joki et al., 2001; Muller et al., 1999; Read et al., 2001). The purpose of this study was to develop a method for pre- paring monodisperse mammalian cell-enclosing subsieve- sized microcapsules, <100 Am in diameter, from viscous polymer solutions. The reduction in diameter enhances cell viability due to reduced resistance to the transport of oxygen, nutrients, and metabolites. Mechanical strength (Ross and Chang, 2002) and biocompatibility (Robitaille et al., 1999) are also enhanced. In addition, the reduction in diameter increases the alternatives for implantation sites. One of the well-known methods for producing subsieve-sized capsules is the emulsification technique by mixing with a magnetic stirrer and a homogenizer (Lim et al., 2000; Ribeiro et al., 1999). However, it is difficult to obtain monodisperse cap- sules. In addition, adding shear stresses and sonicating for mixing are harmful to mammalian cells. Recently, it has been reported that monodisperse drops can be obtained by the drop breakup technique in a coflowing liquid stream (Ganan-Calvo and Gordillo, 2001; Umbanhowar et al., 2000). In this method, drops form at the tip of a capillary and then detach when they reach a size where the drag force exerted by the coflowing stream exceeds the interfacial tension (Umbanhowar et al., 2000). The detached drops form B 2004 Wiley Periodicals, Inc. Correspondence to: Shinji Sakai