Biomaterials 28 (2007) 3656–3667 Transient inter-cellular polymeric linker Siew-Min Ong a,c,1 , Lijuan He b,d,1 , Nguyen Thi Thuy Linh e , Yee-Han Tee f , Talha Arooz a , Guping Tang a,g , Choon-Hong Tan b,e,ÃÃ , Hanry Yu a,b,c,f,h,i,j,Ã a Institute of Biotechnology and Nanotechnology, A*STAR, The Nanos, No. 04-01, 31 Biopolis Way, Singapore 138669, Singapore b Graduate Program in Bioengineering, Centre for Life Sciences (CeLS), No. 05-01, 28 Medical Drive, Singapore 117456, Singapore c NUS Graduate School for Integrative Sciences and Engineering, Centre for Life Sciences (CeLS), No. 05-01, 28 Medical Drive, Singapore 117456, Singapore d Department of Material Science and Engineering, Johns Hopkins University, Baltimore, MD 21218, USA e Department of Chemistry, Faculty of Science, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore f Department of Physiology, Yong Loo Lin School of Medicine, Level 2 Clinical Research Centre (MD11), 10 Medical Drive, Singapore 117597, Singapore g Department of Chemistry, Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Zhejiang Province 310028, China h Singapore-MIT Alliance, E4-04-10, 4 Engineering Drive 3, Singapore 117576, Singapore i NUS Tissue-Engineering Programme, DSO Labs, National University of Singapore, Singapore 117597, Singapore j Department of Haematology-Oncology, National University Hospital, Singapore 119074, Singapore Received 9 February 2007; accepted 27 April 2007 Available online 3 May 2007 Abstract Three-dimensional (3D) tissue-engineered constructs with bio-mimicry cell–cell and cell–matrix interactions are useful in regenerative medicine. In cell-dense and matrix-poor tissues of the internal organs, cells support one another via cell–cell interactions, supplemented by small amount of the extra-cellular matrices (ECM) secreted by the cells. Here we connect HepG2 cells directly but transiently with inter-cellular polymeric linker to facilitate cell–cell interaction and aggregation. The linker consists of a non-toxic low molecular-weight polyethyleneimine (PEI) backbone conjugated with multiple hydrazide groups that can aggregate cells within 30 min by reacting with the aldehyde handles on the chemically modified cell-surface glycoproteins. The cells in the cellular aggregates proliferated; and maintained the cortical actin distribution of the 3D cell morphology while non-aggregated cells died over 7 days of suspension culture. The aggregates lost distinguishable cell–cell boundaries within 3 days; and the ECM fibers became visible around cells from day 3 onwards while the inter-cellular polymeric linker disappeared from the cell surfaces over time. The transient inter-cellular polymeric linker can be useful for forming 3D cellular and tissue constructs without bulk biomaterials or extensive network of engineered ECM for various applications. r 2007 Elsevier Ltd. All rights reserved. Keywords: PEI-hydrazide; Cell–ECM interaction; Cell aggregates; Tissue engineering; Organ printing; Collagen assay 1. Introduction Three-dimensional (3D) cell cultures are important as they mimic the cell–cell and cell–matrix interactions in tissues in vivo [1]. Classical tissue engineering approaches involve the use of macroscopic polymeric scaffolds for seeding cells, in which the cells are essentially attached to the 2D or pseudo-3D surfaces of the scaffolds. To culture cells in 3D, hydrogels of various kinds have been employed to encapsulate cells [2–4]. These bulk biomaterials might not be appropriate for engineering constructs of cell-dense and matrix-poor tissues of the internal organs [5]. For construction of cellular and tissue structures with minimal or reduced use of extra-cellular matrices (ECM) biomater- ials, cell-conforming ECM or micro-/nano-scale scaffolds have been used [6,7]. Another approach is to mimic the ARTICLE IN PRESS www.elsevier.com/locate/biomaterials 0142-9612/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2007.04.034 ÃÃ Also corresponding author. Ã Corresponding author. Department of Physiology, Yong Loo Lin School of Medicine, Level 2 Clinical Research Centre (MD11), 10 Medical Drive, Singapore 117597, Singapore. Tel.: +65 65163466; fax: +65 68727150. E-mail addresses: chmtanch@nus.edu.sg (C.-H. Tan), phsyuh@nus.edu.sg (H. Yu). 1 These authors contribute equally to this work.