Surface-Treatment-Induced Three-Dimensional Capillary Morphogenesis in a Microfluidic Platform By Seok Chung, Ryo Sudo, Ioannis K. Zervantonakis, Tharathorn Rimchala, and Roger D. Kamm* Angiogenesis and capillary morphogenesis denote the growth of microvessel sprouts, which has an essential role in development, reproduction, and repair, but also occurs in tumor formation and in a variety of diseases. [1] Since the first in vitro angiogenesis model waspresentedbyFolkman&Haudenschild,manyhaveattemptedto develop assays that better mimic the true in vivo situation. [2] Recent research efforts that are described in review references [3] have provided new insights into the cellular and molecular mechanisms ofangiogenesis, [4] asforexample,intermsoftheroleofrecombinant humanvascularendothelialgrowthfactor(VEGF) [5] andmechanical stresses. [6] However, there remains a need for quantitative angiogenic assays [3a] and 3D models [7] providing a more physio- logically relevant microenvironment of living tissues in vivo compared to previous 2D culture models. [8] Tissue engineering applications used to restore, maintain orenhance tissuesand organs further require a better understanding of 3D structures and responses of cells. [9] To meet the needs for 3D models, assays adapting hydrogel scaffolds, [10] stack-up method [11] or endothelial cell plated beads [12] were introduced. However, these studies could not produce well controlled microenvironments with dimensions similar to the relevant tissue structures in vivo. [13] The continuing need for technologies that recognize, quantify and perturb localized cellular morphogenesis was also expressed in a recent study of cell migration. [14] Griffith and Swartz suggested using microfluidic approaches for capturing complex 3D tissue physiology and mimicking in vivo conditions in vitro. [15] Microfluidics offer the possibility of applying spatially and temporary controlled gradients of chemical factors in culture and quantifying their responses. [3c] Many research groups have presented robust assays to investigate migration of neutrophils, leukemia cells, stem cells, bacteria and cancer cells under varying chemical gradients in microfluidic channels. [16] These studies applied controlled chemical gradients to the cells in a microfluidic channel, reporting the responses of cells migrating on the channel surface or suspended in medium. In other studies, hydrogel scaffolds were cast in microfluidic channels in order to mimic the extracellular matrix (ECM). Molecular diffusion across these scaffolds was analyzed under flow conditions [17] and cells, i.e., neutrophils, hepatocytes, carcinoma cell lines, astrocytes, and macrophage-like cells, [16e,18] were cultured within the hydrogel scaffold under linear chemical gradients to study their migration. [13,16b–e] Kim et al. and Toh et al. also reported that hepatocytes formed 3D structures when they were mixed and cultured within a hydrogel scaffold. [18a,b] Three-dimensional capillary morphogenesis into these scaffolds mimicking in vivo behavior, however, has not yet been realized in a microfluidic platform, nor has the sprouting of endothelial cells into hydrogel scaffold forming in vivo like 3D capillary structures. (The different modes of cell migration previously observed in microfluidic platforms are categorized in Supporting Informa- tion Table 1.) Microfluidic devices made of hydrogel can be expected to induce 3D responses of the cells seeded in the channel into the surrounding hydrogel. [19] However, they reported limited cellular morphogenesis into the hydrogel due to handling difficulties and the need for the hydrogel to serve as the primary structural material of the device. We previously reported the formation of vascular networks inside a collagen scaffold between microfluidic channels. [20] One aspect of these structures, however, was non-physiologic. Rather than forming vessels directly, the endothelial cells plated on the walls of the channel or gel often migrated into the gel as a sheet and initially remained attached to the side walls of the gel chamber (Supporting Information Table 1; 2.5D, migrating cells). These planar structures eventually formed lumens and bridged across the gel region but the process by which these networks formed, initially as sheets adherent to an artificial channel surface, differs from the process of capillary network formation in vivo. Here we introduce a new surface treatment for the microfluidic platform to induce physiologically relevant 3D capillary morphogenesis. (see Fig. 1a for a schematic of the developed microfluidic concept and Supporting Information Fig. 1 for a schematic of a three-channel microfluidic device used as an example. Device preparation details are provided in the Experimental section and Supporting Information Fig. 2.) Endothelial cells were seeded and cultured in one microfluidic channel (cell channel) in direct contact with hydrogel scaffolds. After 12 h, a continuous endothelial monolayer (EC monolayer) formed in the cell channel (Supporting Information Fig. 3) and a growth factor gradient was established from the condition channel to induce the cells to migrate toward the opposing channels through the scaffolds, more on the condition side than on the control side. We show here that surface treatment on the COMMUNICATION www.advmat.de [*] Prof. R. D. Kamm, I. K. Zervantonakis, T. Rimchala Department of Mechanical Engineering and Department of Biologi- cal Engineering Massachusetts Institute of Technology Cambridge, MA 02139 (USA) E-mail: rdkamm@mit.edu Prof. S. Chung School of Mechanical Engineering, Korea University Anam-Dong, Seongbuk-Gu, Seoul 136-713 (Korea) Prof. R. Sudo Department of System Design Engineering, Keio University 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522 (Japan) DOI: 10.1002/adma.200901727 Adv. Mater. 2009, 21, 4863–4867 ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4863