MICROFLUIDIC FABRICATION OF CELL AND TISSUE ARCHITECTURE Shoji Takeuchi 1,2,3

1Institute of Industrial Science, University of Tokyo, 2Kanagawa Academy of Science and Technology and

3ERATO, JST, JAPAN ABSTRACT The microfluidic technology has progressed remarkably with a variety of applications in analytical chemistry, cell biology, point of care diagnosis and particle handling [1]. This technology is, I think, also powerful for the construction of miniaturized structure composed of biological components. In this talk, I will discuss how we can reconstruct the biological architectures such as cellular membrane and 3D tissue-like structure by using the laminar-flow or droplet microfluidic technology.

Keywords: artificial lipid bilayers, monodisperse liposomes, cell fibers, bottom-up tissue engineering

ARTIFICIAL CELL FABRICATION

Artificial cellular membrane incorporating different membrane proteins can be used for a variety of purposes, including next-generation diagnosis, drug discovery, and highly- sensitive ion-channel-based biosensors [2].

Our group developed a reproducible method to form a planar lipid bilayer without apertures using simple fluidic control: droplet contacting method (Fig. 1a) [3]. At the interface between water and organic solvent containing amphipathic molecules (phospholipids), the monolayer assembles spontaneously. Once the two interfaces come into contact with each other, they immediately form a lipid bilayer. This system was found to be stable, and formation of multiple membranes is readily achieved.

For the ion channel recording with this membrane, we used human BK ion channels [4] or alpha hemolysins [5], and achieved parallel single ion channel recording (Fig. 1b), indicating that this membrane can be used for the electrophysiological study of membrane proteins.

Using this lipid bilayer, we have developed a method for the preparation of lipid vesicles inspired from the formation of soap bubbles from a soap film (Fig. 1c) [6, 7]. In this method, lipid vesicles are blown out of the planar lipid membrane, directly encapsulating ejected materials. This method allows rapid preparation of uniformly sized vesicles, without post-processing.

Such vesicle formation technology might be useful in medical and biological applications, such as encapsulating containers for biological materials, chemicals, and drugs. Since the monodisperse liposomes provide small reaction volumes similar to those of living cells, they are also primary models for the study of cell systems (Fig. 1d,e); artificial cell studies require vesicles of uniform size with biologically functionalized membranes.

Figure 1: Artificial lipid bilayer formation (a) droplet contact method (b) highly parallel single ion channel recording (c) vesicles formation thru a pulsed jet flow (d, e) highly monodisperse liposome array formed by gentle hydration of patterned lipid films

Figure 2: 3D tissue construction (a) cell laden hydrogel microbeads (b) 3D macroscopic tissue structure constructed by molding the cell beads (c) cell laden microfiber (d) vascular structure induced from a cell fiber (e) primary-islet-cell fiber implanted into a kidney capsule of a diabetic mouse (f) cell laden microplates before folding into 3D structure

978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 1300 17th International Conference on MiniaturizedSystems for Chemistry and Life Sciences27-31 October 2013, Freiburg, Germany

3D TISSUE FABRICATION Recently, “bottom-up” tissue engineering approaches including the method of cell sheets, and inkjet printing of cells

or spheroids has been widely studied. Using these methods, building-up tissue with high density becomes possible. Our group recently developed cell-laden micro beads [8, 9, 10], micro fibers [11, 12] and micro plates [13] as building blocks for the 3D construction; the tissue structures are formed by molding, weaving, and folding of each block.

Monodisperse collagen gel beads are prepared by a droplet microfluidic technology [10], and the size-controlled cell beads are obtained by seeding cells over the collagen gel beads or by encapsulating the cells (Fig. 2a). The cell beads are then molded into the designed silicone chamber to form macroscopic 3D tissue structure (Fig. 2b). The bead-stacking structure allows nutrients to reach cells located in the center of the tissue, preventing necrosis during tissue formation for more than a day. This approach enables the rapid and reproducible construction of large-scale 3D tissues with a complex microstructure.

We also developed meter-long gel microfibers that encapsulate cells and extracellular-matrix proteins and can replicate intrinsic functionalities of tissues [11]. The microfibers can be woven and reeled into tissue-like shapes. Using a double-coaxial microfluidic device, we embedded cells in natural extracellular-matrix proteins and protected them with a rapidly gelating hydrogel shell (Fig. 2c). We found that the resulting fibers beat spontaneously when we contained cardiomyocytes, and that tubular structure (Fig. 2d) or neural networks formed in the fibers when we encapsulated endothelial cells or cerebral cortical cells. We also showed that microfibers of pancreatic islet cells transplanted underneath the kidney of diabetic mice normalize the concentration of glucose (Fig. 2e); these fibers can later be removed.

Regarding cell laden microplates, we found that two micro plates can be lifted-up by cell-traction force once the cells are extended across two adjacent micro plates (Fig. 2f); immediately after detaching the plates from the substrate by a micromanipulator, the plates were lifted and folded up into 3D structures due to the traction forces caused by stretched cells between two plates [13]. We used this effect to form 3D hollow tissue structures. In our preliminary experiments, we successfully produced 3D micro structures in various shapes such as cube, dodecahedron and tube structures using 3T3 cells. REFERENCES [1] W-H. Tan and Shoji Takeuchi: A Trap-and-Release Integrated Microfluidic System for Dynamic Microarray Appli-

cations, Proc. Natl. Acad. Sci. USA, vol. 104, no. 4, pp. 1146-1151, 2007 [2] N. Misawa, H. Mitsuno, R. Kanzaki, S. Takeuchi: A Highly Sensitive and Selective Odorant Sensor using Living

Cells Expressing Insect Olfactory Receptors, Proc. Natl. Acad. Sci. USA, vol. 107(35), pp. 15340-15344, 2010 [3] K. Funakoshi, H. Suzuki, and S. Takeuchi: Lipid bilayer formation by contacting monolayers in a microfluidic de-

vice for membrane protein analysis, Analytical Chemistry, vol. 78, pp. 8169-8174, 2006. [4] R. Kawano, Y. Tsuji, K. Sato, T. Osaki, K. Kamiya, M. Hirano, T. Ide, N. Miki, and S. Takeuchi: Automated Paral-

lel Recordings of Topologically Identified Single Ion Channels, Scientific Reports, 3: 1995|DOI: 10.1038 /srep01995 [5] R. Kawano, T. Osaki, H. Sasaki, M. Takinoue, S. Yoshizawa and S. Takeuchi: Rapid Detection of a Cocaine-

Binding Aptamer Using Biological Nanopores on a Chip, J. Am. Chem. Soc., vol. 133, no. 22, pp 8474-8477, 2011 [6] K. Funakoshi, H. Suzuki, and S. Takeuchi: Formation of giant lipid vesicle-like compartments from a planar lipid

membrane by a pulsed jet flow, J. Am. Chem. Soc., vol. 129, pp. 12608-12609, 2007 [7] S. Ota, S. Yoshizawa, and S. Takeuchi, Microfluidic Formation of Monodisperse, Cell-sized and Unilamellar Vesi-

cles, Angew. Chem. Int. Ed., vol. 48, pp. 6533-6537, 2009 [8] W-H. Tan and S. Takeuchi: Monodisperse Alginate Hydrogel Microbeads for Cell Encapsulation, Advanced Materi-

als, vol. 19, pp. 2696-2701, 2007 [9] K. Maeda, H. Onoe, M. Takinoue, and S. Takeuchi: Controlled synthesis of 3D multi-compartmental particles with

centrifuge-based microdroplet formation from a multi-barrelled capillary, Advanced Materials, vol. 24(10), pp. 1340-1346, 2012

[10] Y. Matsunaga, Y. Morimoto and S. Takeuchi, Bead-based tissue engineering: moulding cell beads into a 3D tissue architecture, Advanced Materials, vol. 23, no.12, pp. H90-H94, 2011

[11] H. Onoe, T. Okitsu, A. Itou, M. Kato-Negishi, R. Gojo, D. Kiriya, K. Sato, S. Mirua, S. Iwanaga, K. Kuribayashi-Shigetomi, Y. Matsunaga, Y. Shimoyama, and S. Takeuchi: Metre-long Cellular Microfibres Exhibit Tissue Mor-phologies and Functions, Nature Materials, vol.12, pp. 584–590, 2013

[12] K. Hirayama, T. Okitsu, H. Teramae, D. Kiriya, H. Onoe and S. Takeuchi: Cellular building unit integrated with mi-crostrand-shaped bacterial cellulose, Biomaterials, vol. 34, pp. 2421-2427, 2013

[13] K. Kuribayashi-Shigetomi, H. Onoe, and S. Takeuchi: Cell Origami: Self-folding of Three-Dimensional Cell-Laden Microstructures Driven by Cell Traction Force, PLOS ONE, vol. 7(12), p. e51085, 2012

CONTACT *Shoji Takeuchi, tel: +81-3-5452-6650; takeuchi@iis.u-tokyo.ac.jp

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