Controlled 3D culture in Matrigel microbeads to analyze clonal acinar development.

3D culture systems are a valuable tool for modeling morphogenesis and carcinogenesis of epithelial tissue in a structurally appropriate context. We present a novel approach for 3D cell culture based on a flow-focusing microfluidic system that encapsulates epithelial cells in Matrigel beads. As a model we use prostatic and breast cells and assay for development of acini, polarized cellular spheres enclosing lumen. Each individual bead on average acts as a single 3D cell culture compartment generating one acinus per bead. Compared to standard protocols microfluidics provides increased control over the environment leading to more a uniform acini population. The increased facility of bead manipulation allowed us to isolate single cells which are self-sufficient to fully develop into acini in presence of Matrigel. Furthermore, combination of our microfluidic approach with large particle FACS opens new avenues in high throughput screening on single acini or spheroids.

[1]  D. Weitz,et al.  Single-cell analysis and sorting using droplet-based microfluidics , 2013, Nature Protocols.

[2]  H. Stone,et al.  Formation of dispersions using “flow focusing” in microchannels , 2003 .

[3]  P. Lee,et al.  Microfluidic array for three-dimensional perfusion culture of human mammary epithelial cells , 2011, Biomedical microdevices.

[4]  M. Webber,et al.  Laminin‐1 and α6β1 integrin regulate acinar morphogenesis of normal and malignant human prostate epithelial cells , 2001 .

[5]  Kenneth M. Yamada,et al.  Taking Cell-Matrix Adhesions to the Third Dimension , 2001, Science.

[6]  A. Khademhosseini,et al.  Microscale technologies for tissue engineering and biology. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Matthias P Lutolf,et al.  Biomaterials meet microfluidics: building the next generation of artificial niches. , 2011, Current opinion in biotechnology.

[8]  Helen Song,et al.  Reactions in droplets in microfluidic channels. , 2006, Angewandte Chemie.

[9]  J. S. Johnson,et al.  Biocompatible surfactants for water-in-fluorocarbon emulsions. , 2008, Lab on a chip.

[10]  G. Lajoie,et al.  Matrigel: A complex protein mixture required for optimal growth of cell culture , 2010, Proteomics.

[11]  K. Mostov,et al.  From cells to organs: building polarized tissue , 2008, Nature Reviews Molecular Cell Biology.

[12]  Valerie M. Weaver,et al.  A tense situation: forcing tumour progression , 2009, Nature Reviews Cancer.

[13]  K. Oh,et al.  Generation of core-shell microcapsules with three-dimensional focusing device for efficient formation of cell spheroid. , 2011, Lab on a chip.

[14]  A. van den Berg,et al.  High-yield cell ordering and deterministic cell-in-droplet encapsulation using Dean flow in a curved microchannel. , 2012, Lab on a chip.

[15]  M. Webber,et al.  Acinar Differentiation by Non-malignant Immortalized Human Prostatic Epithelial Cells and Its Loss by Malignant Cells Expression of the Basement Membrane Protein Laminin, Cell Adhesion Molecules Such as Cadherins and Integrin Receptors for Extracellular Matrix (ecm) Proteins. Normal Expression Of , 2022 .

[16]  D. Weitz,et al.  Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity. , 2009, Lab on a chip.

[17]  Mary Kay Harper,et al.  3D Models of Epithelial-Mesenchymal Transition in Breast Cancer Metastasis , 2011, Journal of biomolecular screening.

[18]  Jean-Louis Viovy,et al.  Microfluidic high-throughput encapsulation and hydrodynamic self-sorting of single cells , 2008, Proceedings of the National Academy of Sciences.

[19]  Yanan Du,et al.  Micro-scaffold array chip for upgrading cell-based high-throughput drug testing to 3D using benchtop equipment. , 2014, Lab on a chip.

[20]  Shoji Takeuchi,et al.  Encapsulating bacteria in agarose microparticles using microfluidics for high-throughput cell analysis and isolation. , 2011, ACS chemical biology.

[21]  W J Nelson,et al.  Steps in the morphogenesis of a polarized epithelium. II. Disassembly and assembly of plasma membrane domains during reversal of epithelial cell polarity in multicellular epithelial (MDCK) cysts. , 1990, Journal of cell science.

[22]  Florian Hollfelder,et al.  Microfluidic droplets: new integrated workflows for biological experiments. , 2010, Current opinion in chemical biology.

[23]  P. Garstecki,et al.  Iterative operations on microdroplets and continuous monitoring of processes within them; determination of solubility diagrams of proteins. , 2012, Lab on a chip.

[24]  Jayanta Debnath,et al.  Modeling morphogenesis and oncogenesis in three-dimensional breast epithelial cultures. , 2008, Annual review of pathology.

[25]  B. Weigelt,et al.  The need for complex 3D culture models to unravel novel pathways and identify accurate biomarkers in breast cancer. , 2014, Advanced drug delivery reviews.

[26]  Jeffrey A Hubbell,et al.  Biomaterials science and high-throughput screening , 2004, Nature Biotechnology.

[27]  Joanna F. Pearson,et al.  Polarised fluid movement, and not cell death creates luminal spaces in adult prostate epithelium , 2008, Cell Death and Differentiation.

[28]  Shoji Takeuchi,et al.  Molding Cell Beads for Rapid Construction of Macroscopic 3D Tissue Architecture , 2011, Advanced materials.

[29]  J. Brugge,et al.  Lumen formation during mammary epithelial morphogenesis: insights from in vitro and in vivo models , 2008, Cell cycle.

[30]  D. Beebe,et al.  The present and future role of microfluidics in biomedical research , 2014, Nature.

[31]  Andrew D Griffiths,et al.  Droplet-based microfluidic systems for high-throughput single DNA molecule isothermal amplification and analysis. , 2009, Analytical chemistry.

[32]  N. Perrimon,et al.  Droplet microfluidic technology for single-cell high-throughput screening , 2009, Proceedings of the National Academy of Sciences.

[33]  Brian W. Pogue,et al.  An imaging-based platform for high-content, quantitative evaluation of therapeutic response in 3D tumour models , 2014, Scientific Reports.

[34]  Monpichar Srisa-Art,et al.  Microdroplets: a sea of applications? , 2008, Lab on a chip.

[35]  D. Ornstein,et al.  Culture requirements of prostatic epithelial cell lines for acinar morphogenesis and lumen formation in vitro: Role of extracellular calcium , 2007, The Prostate.

[36]  P. Garstecki,et al.  Discontinuous transition in a laminar fluid flow: a change of flow topology inside a droplet moving in a micron-size channel. , 2012, Physical review letters.

[37]  C. Lovitt,et al.  Miniaturized three-dimensional cancer model for drug evaluation. , 2013, Assay and drug development technologies.

[38]  Keith E. Mostov,et al.  Building epithelial architecture: insights from three-dimensional culture models , 2002, Nature Reviews Molecular Cell Biology.

[39]  H. Kleinman,et al.  Matrigel: basement membrane matrix with biological activity. , 2005, Seminars in cancer biology.

[40]  Cynthia A. Reinhart-King,et al.  Tensional homeostasis and the malignant phenotype. , 2005, Cancer cell.

[41]  Ales Prokop,et al.  NanoLiterBioReactor: Long-Term Mammalian Cell Culture at Nanofabricated Scale , 2004, Biomedical microdevices.

[42]  M. Webber,et al.  Laminin-1 and alpha6beta1 integrin regulate acinar morphogenesis of normal and malignant human prostate epithelial cells. , 2001, The Prostate.

[43]  H. Kleinman,et al.  Role of collagenous matrices in the adhesion and growth of cells , 1981, The Journal of cell biology.

[44]  Min Jun Kim,et al.  Microfluidics-generated pancreatic islet microfibers for enhanced immunoprotection. , 2013, Biomaterials.

[45]  Genee Y. Lee,et al.  Three-dimensional culture models of normal and malignant breast epithelial cells , 2007, Nature Methods.

[46]  D. Chiu,et al.  Droplets for ultrasmall-volume analysis. , 2009, Analytical chemistry.

[47]  G. Cunha,et al.  Morphogenesis of ductal networks in the mouse prostate. , 1986, Biology of reproduction.

[48]  Anirban Datta,et al.  Epithelial polarity and tubulogenesis in vitro. , 2003, Trends in cell biology.

[49]  J. Kawamura,et al.  Morphological and functional heterogeneity in the rat prostatic gland. , 1991, Biology of reproduction.

[50]  Tanyu Wang,et al.  Quantitative microfluidic biomolecular analysis for systems biology and medicine , 2013, Analytical and Bioanalytical Chemistry.

[51]  Jayanta Debnath,et al.  Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. , 2003, Methods.

[52]  Haakan N. Joensson,et al.  Droplet Microfluidics — A Tool for Single‐Cell Analysis , 2013 .

[53]  D. Fletcher,et al.  Patterned Collagen Fibers Orient Branching Mammary Epithelium through Distinct Signaling Modules , 2013, Current Biology.

[54]  M. Théry,et al.  Micropatterning as a tool to decipher cell morphogenesis and functions , 2010, Journal of Cell Science.