3D Cell Culturing with the Bio-Assembler from Nano3D Biosciences, Inc.

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1 A Nano3D Biosciences White Paper 7000 Fannin St., suite 2140 Houston, TX D Cell Culturing with the Bio-Assembler from January 2012 By Glauco R. Souza, Ph.D. (CSO) and Thomas C. Killian, Ph.D. (Founder and Professor of Physics and Astronomy, Rice University)

Nano3D Biosciences presents the Bio-Assembler TM, the next generation of cell culturing technology,withthe following advantages or unique features for basic research, toxicology, drug discovery, and

Compared to traditional, two-dimensional (2D) cell culture on plastic, the Bio- Assembler results in: o Accurate reproduction of the three-dimensional (3D) microenvironment of in vivo tissue o

2 Nano3D Biosciences presents the Bio-Assembler TM, the next generation of cell culturing technology,withthe following advantages or unique features for basic research, toxicology, drug discovery, and regenerative medicine. Compared to traditional, two-dimensional (2D) cell culture on plastic, the Bio- Assembler results in: o Accurate reproduction of the three-dimensional (3D) microenvironment of in vivo tissue o Representative in vivocell morphology, protein expression, and response to exogenous agents Compared to other3d cell culture platforms, the Bio-Assembler provides: o 10x faster setup and creation of 3D structures and cell-cell interactions o Compatibility with any cell type including primary and stem cells o Compatibility with any media type, standard culturing protocols, and diagnostic techniques o Compatibility with high-throughput techniques o 3D co-culturing capabilities o Unique capabilities for moving and shaping tissue, for applications such as invasion studies and tissue engineering o Tissue self assembly and endogenous extracellular matrix creation o No exogenous extracellular matrix o No lot-to-lot variability o No expensive equipment required * Contact us through to bring this exciting new technology to your research today. 2

3 Table of Contents Overview Technical Notes I. 3D cell culturing by magnetic levitation with the Bio-Assembler II. Applications and Assays Using 3D Cell Culturing by Magnetic Levitation III. Biocompatibility of Nanoparticles References

4 3D cell culture with the Bio-Assembler. Pulmonary fibroblasts cultured with the Bio-Assembler. Immunohistochemistry of levitated human glioblastoma cells showing in vivo-like N-cadherin expression. Overview. Standard monolayer cell culturing on tissue culture plastic has notably improved our understanding of basic cell biology, but it does not replicate the complex 3D architecture of in vivo tissue, and it can significantly modify cell properties. This often compromises experiments in basic life science, leads to misleading drug-screening results on efficacy and toxicity, and produces cells that may lack the characteristics needed for developing tissue regeneration therapies 1-8. The future of cell culturing for fundamental studies and biomedical applications lies in the creation of multicellular structure and organization in three-dimensions 3,4,6,7,9-11. Many schemes for 3D culturing are being developed or marketed, such as bio-reactors 12 or protein-based gel environments 6,13, but they suffer from high cost, low- or the presence throughput, poor scalability, complexity, of non-human biological factors that can alter cell behavior and preclude therapeutic use 6, The magnetic levitation method, marketed as the Bio- in cell culturing Assembler, 1 is a new paradigm developed by Nano3D Biosciences that provides the advantages of 3D cell culturing in a platform that is no more complicated than standard 2D culturing. The Bio- with existing Assembler can easily be incorporated protocols and diagnostics such as in situ imaging, fixing, and immunohistochemistry. The Bio-Assembler uses biocompatible polymer-based reagents to deliver magnetic nanoparticles to individual cells so that an applied magnetic drive can levitate cells off the bottom of the culture dish and bring cells together near the air-liquid interface. This initiates cell-cell interactions in the absence of any artificial surface or matrix. Magnetic fields are designed to rapidly form 3D multicellular structures in as little as a few hours, including expression of extracellular matrix proteins, which is much faster than any other 3D cell culturing technique. The morphology, protein expression, and response to exogenous agents of resulting tissue show great similarity to in vivo results. 4

5 There is no need for expensive equipment or special media or growth factors, and the ability to manipulate cells and shape tissue magnetically offers new possibilities for controlled co-culturing and invasionn assays. This powerful technology is optimized for use in basic research and applications in toxicology, drug discovery, stem cell studies, and tissue engineering. Fibronectin expression in levitated dental pulp stem cells. Levitated neural stem cells. All cell types that have been tested with the Bio- including Assembler have been cultured successfully, human cell lines (glioblastomas, normalastrocytes, HEK 293, breast cancermda-231, breast MCF10A, 3T3- fibrolast - pre-adipocytes, hepatoma H-4-II-E, bone marrow endothelial cells, melanoma, Kaposi s sarcoma), stem cells (murine neural stem cells, mesenchymal stem cells, and dental pulp stem cells), and primary cells (endothelial, smooth muscle, epithelial, fibroblasts, chondrocytes, and cells isolated from adipose). The Bio-Assembler will bring the benefits of 3D cell culturing to your life science applications with negligible increase in experimental complexity and without the need for any expensive equipment. Customers and collaborators are currently using the Bio-Assembler in a wide range of fields. Examples includenephrotoxicity assays, drug efficacy assays, brain cancer invasion assays, creation of layered lung tissue co-cultures for studies of lung remodeling, and creation of organ-like 3D cultures (organoids) with stem cells. The following pages provide a schematic of the method and selected results and assays using the Bio-Assembler. The biocompatibility of nanoparticles is also discussed. Acinar structure in mammary epithelial cells grown with the Bio-Assembler. Contact us through n3dbio.com or info@n3dbio.com to learn how simple it is to bring this exciting new technology to your laboratory. 5

6 3D Cell Culturing with the Bio-Assembler from Technical Notes. I. 3D cell culturing by magnetic levitation with the Bio-Assembler. The Bio-Assembler is as simple as standard 2D cell culturing, and it is compatible with all cell types, media, and diagnostics. Fig. 1. 3D cell culturing through magnetic levitation with the Bio-Assembler. (A) Nanoshuttle containing magnetic nanoparticles is added and dispersed over cells and the mixture is incubated. (B) After incubation with Nanoshuttle, cells are detached and transferred to a petridish. (C) A magnetic drive is then placed on top of a petri dish top. (D) The magnetic field causes cells to rise to the air medium interface. (E) Human umbilical vein endothelial cells (HUVEC) levitated for 60 minutes (left images) and 4 hours (right images) (Scale bar, 50 µm).the onset of cell-cell interaction takes place as soon as cells levitate, and 3D structures start to form within minutes of levitation. At 1 hour, the cells are still relatively dispersed, but they are already showing some signs of stretching. Formation of 3D structures is visible after 4 hours of levitation (arrows). 6

7 Morphology is a good baseline metric for cell cultures, especially for assessing in vivo similarity. 15 In levitated cultures, we observe morphology characteristic of in vivo tissue, which we attribute to the absence of a perturbing plastic surface and the ability of cells to interact and expand in a more natural 3D microenvironment of other cells and signals. Fig.2. (A) Human primary alveolar epithelial cells cultured by magnetic levitation at the air-liquid interface (30 µm scale bar) spontaneously assemble in layers with a "tile-like" squamous morphology that is difficult to produce in vitro and resembles squamous epithelial lung tissue (Right). (B) Mammary epithelial cells grown with magnetic levitation (Left) develop characteristic acinar structures, which are difficult to achieve in 2D and are indicative of normall cell polarization and growth arrest and can allow one to distinguish between normal and tumorigenic cells 16. Results are very similar to observations in the current 3D culture standard - reconstituted basement membrane (Matrigel ). Fig.3. Human umbilical vein endothelial cells (HUVEC) are often the cells of choice for in vitro models for angiogenesis. In approximately 12 hours, HUVEC cells cultured with the Bio-Assembler TM (Left, scale bar, 100µm) form characteristic sprouting structure reminiscent of in vivo and matching results obtained with Matrigel (Middle), otocol/ph1212. (Right) Photograph of macrostructure of cells cultured for 24 hours (scale bar, 5 mm; magnet was removed prior to taking photograph). Protein expression in levitated cultures shows striking similarity to in vivo patterns. N- cadherin expression in levitated human glioblastoma cells was identical to the expression seen in human tumor xenografts grown in immunodeficient mice, while standard 2D culture showed 7

8 much weaker expression that did not match xenograft distribution 1 (Fig. 4). The transmembrane protein N-cadherin is often used as an indicator of in-vivo-like tissue assembly in 3D culturing 1. Fig.4. Distribution of N-cadherin (red) and nuclei (blue) in human brain cancer mouse xenograft (left, human brain cancer cells grown in a mouse brain), brain cancer cells cultured by 3D magnetic levitation for 48 h. (middle), and cells cultured on a glass slide cover slip (2D, right). The 2D system shows N-cadherin in the cytoplasm and nucleus and notably absent from the membrane, while in the levitated culture and mouse, N-cadherin is clearly concentrated in the membrane, and also present in cytoplasm and cell junctions. Extracellular Matrix (ECM) Production. All cell types we have cultured to date with magnetic levitation form strong, cohesive tissue structures in less than 24 hrs. 1 The magnetic field enables cell levitation,, but it also guides cells together and promotes rapid cell-cell interaction. 3D cellular assembly takes place without the influence of an artificial ECM. Cells start to generate an endogenous ECM within hours of levitation (Fig. 5). Fig.5: Immunohistochemical staining of extracellular matrix proteins (fibronectin, collagen I, laminin) expressed in magnetically levitated 3D single cell-type levitated cultures in comparison to 2D cultures of human primary fibroblast (HPF). These results show robust ECM formation in 3D. 8

9 II. Applications and Assays Using 3D Cell Culturing by Magnetic Levitation. Co-Culturing, Magnetic Manipulation, and Invasion Assays. The unique ability to manipulate cells and shape tissue magnetically offers new possibilities for controlled co-culturing and invasion assays. Co-culturing in arealistic tissue architecture is critical foraccurately modeling in vivo conditions, such as for increasing the accuracy of cellular assays (Fig. 6). Fig. 6. Invasion assay of magnetically levitated multicellular spheroids. Fluorescence images of human glioblastoma (GBM) cells (green; GFP-expressing cells) and normal human astrocytes (NHA) (red; mcherry-labelled) cultured separately and then magnetically guided together (left, time 0). Invasion of GBM into NHA in 3D culture provides a powerful new assay for basic cancer biology and drug screening (right, 12h to 252h). The invasiveness of levitated glioblastoma cells in normal human astrocytes matched patterns seen in xenograft, while other in vitro culturing methods, including 3D culturing in basement membrane (Matrigel ), failed to reproduce this behavior 2 (Fig. 7). Fig.7: Invasion Assay. Human glioblastoma cells (GBM) grown with the Bio-Assembler. Three-dimensional spheres of GFP-labeled GBM and mcherry-labeled normal human astrocytes (NHAs) were formed separately and then magnetically guided together. Serial fluorescence images show an invading front at the contact area. Less invasive strains of GBM did not display this behavior. 9

10 3D Cell Culturing with the Bio-Assembler from 3D Tissue Reconstruction Assay for Viability, Toxicity, and/or Drug Efficacy: Nephrotoxicity Assay.Cell migration and proliferation diagnostics are useful for toxicity screening and drug disco discovery, very, as these properties correlate with toxicity or efficacy of artificial compounds. Based on the Bio-Assembler, nano3d developed the first 3D tissue reconstruction assay, which should be much more predictive predictive than 2D assays. Figure 8 shows the method and results with hhuman uman embryonic kidney (HEK293) cells testing nephratoxicity of ibuprofen. Fig. 8. 3D Tissue reconstruction assay. (Left) (A) Cells are dispersed and treated with Nanoshuttle, (B) magnetically levitated and concentrated to form 3D tissue, (C) magnetically held to allow a capillary tube to pierce the tissue, and (D) periodically imaged to monitor tissue reconstruction. Microphotographs of HEK293 cells grown by magnetic levitation in 3D (E) before before and (F) after hole placement (scale bar is 500 µm). m). (Right) Hole closure as a function of time (0, 2, 4, 24, 48, and 96 hours) and after treatment with 0, 0.1, 1, and 4 mm ibuprofen in DMSO (scale bar is 500 µm). High concentration degrades cell health and suppresses closure. III. Biocompatibility of Nanoparticles. During levitated culturing, cells uptake magnetic nanoparticles through electrostatic mediated cell membrane adhesion, therefore the effects of the nanoparticles on biological function is an obvious question. The particles used are biocompatible, well tolerated by cells, and commonly used in many biological applications applications,, and we have observed no deleterious effects of nanoparticles in any of the cell types cultured. Cells remain healthy and continue to expand literally for months1, stopping only at cessation of the experiment. TEM images1 show that cells expel nanoparticles into the surrounding extracellular matrix within a few days. Comparative genomic hybridization (CGH) screening of pprimary rimary cells showed no chromosomal abnormalities in cells treated with nanoparticles and levitated. The application of forces to cells, as studied in mechano-biology, can up-regulate protein expression and drive cellular differentiation, but the forces applied to cells are weak compared to forces generated by the cells themselves. Any perturbations caused by nanoparticles and levitation forces are negligible compared to the improved in-vivo similarity offered by 3D culturing. 10

11 References 1 Souza, G. R. et al. Three-dimensional Tissue Culture Based on Magnetic Cell Levitation. Nature Nanotechnol. 5, , doi: /nnano (2010). 2 Molina, J., Hayashi, Y., Stephens, C. & Georgescu, M.-M. Invasive glioblastoma cells acquire stemness and increased Akt activation. Neoplasia 12, (2010). 3 Pampaloni, F., Reynaud, E. G. & Stelzer, E. H. K. The third dimension bridges the gap between cell culture and live tissue. Nat. Rev. Mol. Cell Biol. 8, (2007). 4 Cukierman, E., Pankov, R., Stevens, D. R. & Yamada, K. M. Taking Cell-Matrix Adhesions to the Third Dimension. Science 294, (2001). 5 Abbott, A. Biology's new dimension. Nature 424, (2003). 6 Prestwich, G. D. Simplifying the extracellular matrix for 3-D cell culture and tissue engineering: A pragmatic approach. J. Cell. Biochem. 101, , doi: /jcb (2007). 7 Boudreau, N. & Weaver, V. Forcing the Third Dimension. Cell 125, (2006). 8 Griffith, L. G. & Swartz, M. A. Capturing complex 3D tissue physiology in vitro. Nat. Rev. Mol. Cell Biol.7, (2006). 9 Birgersdotter, A., Sandberg, R. & Ernberg, I. Gene expression perturbation in vitro--a growing case for three-dimensional (3D) culture systems. Semin. Cancer Biol. 15, (2005). 10 Yamada, K. M. & Cukierman, E. Modeling tissue morphogenesis and cancer in 3D. Cell 130, (2007). 11 Atala, A. Engineering tissues, organs and cells. J. Tissue Eng. Regen. Med. 1, (2007). 12 Bilodeau, K. & Mantovani, D. Bioreactors for Tissue Engineering: Focus on Mechanical Constraints. A Comparative Review. Tissue Eng. 12, (2006). 13 Prestwich, G. D., Liu, Y., Yu, B., Shu, X. Z. & Scott, A. 3-D culture in synthetic extracellular matrices: new tissue models for drug toxicology and cancer drug discovery. Adv. Enzyme Regul. 47, (2007). 14 Polykandriotis, E., Arkudas, A., Horch, R. E. & Kneser, U. To matrigel or not to matrigel. Am. J. Pathol. 172, 1441; ,(2008). 15 Sodek, K. L., Brown, T. J. & Ringuette, M. J. Collagen I but not Matrigel matrices provide an MMP-dependent barrier to ovarian cancer cell penetration. BMC Cancer 8, (2008). 16 Wolf, M. Influence of matrigel on biodistribution studies in cancer research. Die Pharmazie 63, (2008). 11

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