Automated microinjection of cell-polymer suspensions in 3D ECM scaffolds for high-throughput quantitative cancer invasion screens Hoa H. Truong a, 1 , Jan de Sonneville b,1 , Veerander P.S. Ghotra a , Jiangling Xiong a , Leo Price a , Pancras C.W. Hogendoorn c , Herman H. Spaink d , Bob van de Water a , Erik H.J. Danen a, * a Division of Toxicology, Leiden Amsterdam Center for Drug Research, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands b Division of Biophysical Structural Chemistry, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands c Department of Pathology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands d Institute of Biology, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands article info Article history: Received 29 August 2011 Accepted 21 September 2011 Available online xxx Keywords: Spheroid Microinjection Screen High-throughput Personalized medicine Cancer abstract Cell spheroids (CS) embedded in 3D extracellular matrix (ECM) serve as in vitro mimics for multicellular structures in vivo. Such cultures, started either from spontaneous cell aggregates or single cells dispersed in a gel are time consuming, applicable to restricted cell types only, prone to high variation, and do not allow CS formation with defined spatial distribution required for high-throughput imaging. Here, we describe a method where cell-polymer suspensions are microinjected as droplets into collagen gels and CS formation occurs within hours for a broad range of cell types. We have automated this method to produce CS arrays in fixed patterns with defined x-y-z spatial coordinates in 96 well plates and applied automated imaging and image analysis algorithms. Low intra- and inter-well variation of initial CS size and CS expansion indicates excellent reproducibility. Distinct cell migration patterns, including cohesive strand-like e and individual cell migration can be visualized and manipulated. A proof-of-principle chemical screen is performed identifying compounds that affect cancer cell invasion/migration. Finally, we demonstrate applicability to freshly isolated mouse breast and human sarcoma biopsy material e indicating potential for development of personalized cancer treatment strategies. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Cells grown under classical 2D culture conditions behave differ- ently from the same cell types grown in vivo. In addition to soluble factors produced in the in vivo microenvironment, differences in cell shape, intercellular contacts, and connections to ECM have striking effects on gene expression, cell survival, proliferation, differentiation, cytoarchitecture, and migration. Various systems have been devel- oped to culture cells within 3D ECM environments, aimed at more closely mimicking the in vivo context [1,2]. Several of these systems produce 3D cell aggregates in which, after compaction, depletion of oxygen, nutrients, and growth factors occurs in the core, leading to cell heterogeneity depending on the position in the resulting cell spheroids (CS) [3,4]. Multistep methods are used in which aggre- gates are allowed to form spontaneously and, following a compac- tion phase, can subsequently be transferred to a 3D ECM. The best- known example of this approach is the “hanging drop assay” that was developed to create embryoid bodies from ES cells and has also been applied to cancer cell lines to produce tumor-like structures [5,6]. Alternative methods involve mixing of single cell suspensions with a solidifying ECM, resulting in individual cells that eventually form CS randomly within a 3D ECM structure [7], or seeding poly- meric scaffolds with cell/ECM suspensions [2]. Cell behavior in 3D cultures is controlled by chemical (compo- sition) and physical (rigidity, cross-linking) properties of the gel. Natural ECM proteins can be used such as collagen, fibrinogen, or the laminin-rich matrigel to represent the in vivo ECM composition most relevant to a given cell type. More recently, synthetic polymers have been developed for 3D CS culture environments although it remains to be established how well these support a variety of cell behavioral outputs, including cell migration [8]. Collagen type 1 is an abundant polymer in ECM in vivo, and it is widely used for 3D cultures. Various physical properties of the collagen gel, such as rigidity and pore size modulate stem cell differentiation, cancer growth, and cell migration [9e11]. Cells can use various migration strategies in 3D environments, including mesenchymal or amoeboid individual cell migration modes or collective invasion strategies, * Corresponding author. Tel.: þ31 71 527 4486. E-mail address: e.danen@lacdr.leidenuniv.nl (E.H.J. Danen). 1 These authors contributed equally. Contents lists available at SciVerse ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2011.09.049 Biomaterials xxx (2011) 1e8 Please cite this article in press as: Truong HH, et al., Automated microinjection of cell-polymer suspensions in 3D ECM scaffolds for high- throughput quantitative cancer invasion screens, Biomaterials (2011), doi:10.1016/j.biomaterials.2011.09.049