Dear All, Below please find the fourteenth issue of the newsletter of the 3D Cell Culture Core (3D3C) Facility of the Birck Nanotechnology Center. The newsletter is also available online (https://nanohub.org/groups/3d3cfacility/news). In this issue, a new section, "3D in action", is added. The five sections in the newsletter are: 3D at Purdue – this section highlights 3D cell culture-based research activity at Purdue 3D in action - this section highlights recent activity in 3D3C 3D in focus – this section presents the current work on a specific 3D cell culture model or technique 3D in publications – this section brings a collection of recent publications on 3D cell culture 3D in meetings – this section includes a list of upcoming meetings related to 3D cell culture The newsletter will be available every two months. If you do not wish to receive the 3D3C newsletter in the future, please reply “cancel” to unsubscribe. Please contact me if you have questions. Yours Sincerely, Tim Kwok Facility Manager 3D Cell Culture Core (3D3C) Facility Birck Nanotechnology Center Purdue University ________________________________ [https://gallery.mailchimp.com/b7d6d1ffee56a866e499104cf/images/3e8e050f-67fb...] Volume 14, February 2018 [https://gallery.mailchimp.com/b7d6d1ffee56a866e499104cf/images/10f15e72-d4d6...] 3D at Purdue 3D in Action 3D in Focus 3D in Publications 3D in Meetings 3D at Purdue Cell-instructive Biomaterials for Musculoskeletal Soft Tissue Regenerative Engineering Regenerative engineering represents a new transdisciplinary paradigm to generate functional tissues and tissue systems based on Convergence of Advanced Materials Science, Stem Cell Science, Physics, Developmental Biology, and Clinical Translation. In particular, biomaterials provide an enabling technology platform to engineer a three-dimensional (3D) scaffold system offering a supportive microenvironment to direct cell migration, growth, differentiation, and organization to form regenerated tissue. The development of 3D scaffolds that mimic the hierarchical architecture of native tissue extracellular matrix requires a fundamental understanding of the structure-function relationship and cell-biomaterial interactions. The Deng Laboratory focuses on both the fundamental understanding of cellular processes in tissue development and engineering and effective biomaterial systems for musculoskeletal regenerative engineering. Our strategies for tissue regeneration embrace the mechanistic understanding of various physicochemical and biological cues on cellular processes, and the application of advanced biomaterials and matrix technologies at the micro- and nanoscale (Figure 1). [https://gallery.mailchimp.com/b7d6d1ffee56a866e499104cf/images/49792c75-b18f...] Figure 1. Design criteria for different material cues on cell-material interactions. Physical cues, such as material topography, stiffness, and porosity, can dramatically affect cell fate and tissue development. Incorporation of chemical cues in the form of simple chemical groups into materials influences cell behavior. Additionally, material carrier presentation of various biological cues such as growth factors and small molecules/inducerons can lead to enhanced cellular responses. Integrating these cues is paramount in creating a synthetic matrix optimized for desirable cellular responses and inductive tissue regeneration (Narayanan, N., Jiang, C., Uzunalli, G., Shalumon, K.T., Laurencin, C.T., Deng, M.: Regenerative Engineering and Translational Medicine. 2016, 2(2):69-84). In our recent collaborative work with Dr. Shihuan Kuang in Animal Sciences, we have developed a bioengineered cell niche composed of aligned polyester fiber scaffolds for skeletal muscle regeneration. Current cell-based therapies for skeletal muscle regeneration are hindered by low survival and long-term engraftment of the transplanted cells due to the lack of appropriate supportive microenvironment (cell niche) in the injured muscle. Therefore, there is a critical need for developing strategies that can provide cellular and structural support for the regeneration of new functional skeletal muscles. Inspired by the native architecture of skeletal muscle, we have created aligned polyester fiber scaffolds mimicking the oriented organization of skeletal muscle by electrospinning using a novel custom-made rotating collector made of parallel metal blades. Aligned fibers with diameters varying from 335 ± 154 nm (nanoscale) to 3013 ± 531 nm (microscale) were fabricated by varying the poly(lactide-co-glycolide) (PLGA) concentration. Myoblasts seeded on aligned PLGA fibers exhibited oriented arrangement according to the direction of fiber alignment resulting from contact guidance (Figure 2). Interestingly, microfiber scaffolds enhanced cell alignment, elongation, proliferation, and differentiation as compared to the nanofiber scaffolds indicating the ability of cells to respond to fiber topography. Pilot in vivo studies were performed to evaluate the regenerative potential of the scaffolds using a dystrophin-deficient mdx mouse model. Following 21 days of implantation, microfiber scaffolds seeded with primary myoblasts resulted in the formation of a network of dystrophin positive myofibers in tibialis anterior muscles of mdx mice. These experiments not only provide critical insights on exploiting the interactions between muscle cells and their microenvironment, but also open new avenues for the development of effective muscle regenerative technologies. [https://gallery.mailchimp.com/b7d6d1ffee56a866e499104cf/images/d8a03b2d-de37...] Figure 2. Fluorescence image of myoblasts on aligned fibers after 24 hours of cell seeding. Cells spread along the direction of fiber orientation on aligned scaffolds (Red: cytoskeletal actin; Blue: nuclei) Relevant Publications: Kumbar, S.G., Laurencin, C.T., Deng, M (Editors): Natural and Synthetic Biomedical Polymers, Elsevier, ISBN: 978-0-12-396983-5, 2014. Narayanan, N., Jiang, C., Uzunalli, G., Shalumon, K.T., Laurencin, C.T., Deng, M.: Polymeric electrospinning for musculoskeletal regenerative engineering. Regenerative Engineering and Translational Medicine. 2016, 2(2):69-84. Funding support from NIH R03AR068108 and Purdue Start-up Package is greatly appreciated. Meng Deng Assistant Professor Department of Agricultural & Biological Engineering ________________________________ 3D in Action Workshop Invitation: 3D3C is invited to organize a workshop for Experimental Biology 2018 Annual Meeting. The workshop is scheduled on April 22, 2018. The title for the workshop is, "The Art of 3D Cell Culture, from Organoids to Organ-on-A-Chip" Recent publication from 3D3C: Lelièvre SA, Kwok T, Chittiboyina S. Architecture in 3D cell culture: An essential feature for in vitro toxicology Toxicol In Vitro. 2017 Dec;45(Pt 3):287-295 Abstract Three-dimensional cell culture has the potential to revolutionize toxicology studies by allowing human-based reproduction of essential elements of organs. Beyond the study of toxicants on the most susceptible organs such as liver, kidney, skin, lung, gastrointestinal tract, testis, heart and brain, carcinogenesis research will also greatly benefit from 3D cell culture models representing any normal tissue. No tissue function can be suitably reproduced without the appropriate tissue architecture whether mimicking acini, ducts or tubes, sheets of cells or more complex cellular organizations like hepatic cords. In this review, we illustrate the fundamental characteristics of polarity that is an essential architectural feature of organs for which different 3D cell culture models are available for toxicology studies in vitro. The value of tissue polarity for the development of more accurate carcinogenesis studies is also exemplified, and the concept of using extracellular gradients of gaseous or chemical substances produced with microfluidics in 3D cell culture is discussed. Indeed such gradients-on-a-chip might bring unprecedented information to better determine permissible exposure levels. Finally, the impact of tissue architecture, established via cell-matrix interactions, on the cell nucleus is emphasized in light of the importance in toxicology of morphological and epigenetic alterations of this organelle. ________________________________ 3D in Focus The architectural and mechanical properties of a scaffold can affect cell behaviors, such as spreading, proliferation and differentiation. Stiffness, a key mechanical property, was studied mostly in systems with a single cell type. Each cell type responds to matrix stiffness in its own way and may affect the other cell types within the same biological system. Subjecting macrophages (Mφ) and bone marrow-derived mesenchymal stem cells (BMMSCs) to a stiffness-tunable matrix, the authors in the highlighted research article investigated how matrix stiffness may regulate the effect of Mφ on osteogenesis of BMMSCS. Xiao-Tao He, Rui-Xin Wu, Xin-Yue Xu, Jia Wang, Yuan Yin, Fa-Ming Chen. Macrophage involvement affects matrix stiffness-related influences on cell osteogenesis under three-dimensional culture conditions. Acta Biomaterialia https://doi.org/10.1016/j.actbio.2018.02.015 3D3C summary of the article: Transglutaminase cross-linked gelatin (TG-gel) with different stiffness levels were prepared by crosslinking different percentages of gelatin with transglutaminase, 3% (Low), 6% (Mid) and 9% (High). The stiffness of TG-gel increased and the porosity decreased with an increase in the percentage of gelatin. Both proliferation and viability of BMMSCs were higher in low-stiffness TG-gels than in high-stiffness TG-gel. The osteogenic potential of the BMMSCs, assessed by Alizarin Red S staining of mineralized nodules, release of alkaline phosphatase (ALP), and the expression levels of osteogenic genes (SP7, OST, RUNX2, ALP and OCN) were elevated with an increase in matrix stiffness. In response to microenvironmental signals, Mφ undergo polarization into distinct functional cells, M1, M2 cells. The polarization status of the Mφ in matrices with different stiffness levels were analyzed via qRT-PCR, immunofluorescence and ELISA. The expression levels of M1-related markers (IL-1β and TNF-α) increased proportionally with an increase in stiffness of the TG-gel. Mφ effects on osteogenesis of BMMSCs cultured in different stiffness levels of TG-gel were examined in a Transwell co-culture system, in which gel-encapsulated Mφ were placed in the upper compartment, and the BMMSCs were in the bottom compartment. The ALP activity and the formation of mineralized nodules in non-encapsulated monolayers of BMMSCs were enhanced when these cells were incubated with Mφ encapsulated in low-stiffness gel compared to high-stiffness gel. Mφ encapsulated in low-stiffness TG-gel increased the formation of mineralized nodules and the ALP activity in BMMSCs encapsulated in either low- or high-stiffness TG-gels, whereas Mφ in high-stiffness TG-gel suppressed these phenomena. By encapsulating Mφ (in the upper compartment) and BMMSCs (in the bottom compartment) in TG-gel of the same stiffness level, the BMMSCs in low- and high-TG-gel displayed higher ALP activity and mineralized nodules formation than the BMMSCSs in mid-stiffness TG-gel, suggesting the nonlinear influence of stiffness for the effect of on BMMSCs osteogenesis. Comments from 3D3C: Extracellular matrix stiffness has profound impacts on cell function and differentiation. Most of the studies related to stiffness are performed, however, on culture systems with a single cell type, while multiple cell types are present in vivo. By encapsulating BMMSCs and Mφ in TG-gel with different stiffness levels in separate chambers of a Transwell system, the studies presented here argue against the conventional rationale that data obtained from a study with a single cell type reflect what happens in vivo where multiple cell types are present. In addition, the possibility that stiffness might vary locally (e.g., next to a particular cell type compared to another cell type) in vivo should also be explored, as results here suggested that the result of the interaction between different cell types might depend on their respective environmental stiffness. An important aspect to explore in future experiment is how the cells would influence each other if placed in the same culture, a system that would better mimics in vivo conditions. ________________________________ 3D in Publications Recent publications on 3D culture (please click to access the list on our web page https://nanohub.org/groups/3d3cfacility): Review<https://nanohub.org/groups/3d3cfacility/news#reviews> The research articles and reviews are arranged in the following categories: Scaffold free/Scaffold Organ/Tissue/Cell Others Spheroids<https://nanohub.org/groups/3d3cfacility/news#spheroids> Organoid<https://nanohub.org/groups/3d3cfacility/news#organoid> Scaffold<https://nanohub.org/groups/3d3cfacility/news#scaffold> Hydrogel<https://nanohub.org/groups/3d3cfacility/news#hydrogel> Matrix<https://nanohub.org/groups/3d3cfacility/news#matrix> Microfluidics<https://nanohub.org/groups/3d3cfacility/news#microfluidic> Microfabrication<https://nanohub.org/groups/3d3cfacility/news#microfabrication> Bone<https://nanohub.org/groups/3d3cfacility/news#bone> Bone Marrow<https://nanohub.org/groups/3d3cfacility/news#bonemarrow> Breast<https://nanohub.org/groups/3d3cfacility/news#breast> Colon<https://nanohub.org/groups/3d3cfacility/news#colon> Heart<https://nanohub.org/groups/3d3cfacility/news#heart> Liver<https://nanohub.org/groups/3d3cfacility/news#liver> Lung<https://nanohub.org/groups/3d3cfacility/news#lung> Muscle<https://nanohub.org/groups/3d3cfacility/news#muscle> Nerve<https://nanohub.org/groups/3d3cfacility/news#nerve> Prostate<https://nanohub.org/groups/3d3cfacility/news#prostate> Endothelial cells<https://nanohub.org/groups/3d3cfacility/news#endothelialcells> Fibroblast<https://nanohub.org/groups/3d3cfacility/news#fibroblast> Stem Cells<https://nanohub.org/groups/3d3cfacility/news#stemcells> Stromal Cells<https://nanohub.org/groups/3d3cfacility/news#stromalcells> Cancer/Tumor<https://nanohub.org/groups/3d3cfacility/news#cancer> Screening<https://nanohub.org/groups/3d3cfacility/news#screening> 3D bioprinting<https://nanohub.org/groups/3d3cfacility/news#3dbioprinting> Imaging<https://nanohub.org/groups/3d3cfacility/news#imaging> ________________________________ 3D in Meetings Keystone Symposia: Organs- and Tissues-on-Chips Date: 8th to 12th April 2018 Location: Big Sky, Montana, USA Website: http://www.keystonesymposia.org/18D1 Contact person:info@keystonesymposia.org Organized by: Keystone Symposia 3rd International Conference on Nanomedicine, Drug Delivery, and Tissue Engineering (NDDTE'18) Date: 10th to 12th April 2018 Location: New Delhi, India Website: http://nddte.com/ Contact person: Conference Administrator Organized by: International ASET 3rd International Conference on 3D Printing in Medicine Date: 4th to 5th May 2018 Location: Halle, Germany Website: http://3dprint-congress.com/ Contact person:congress@bb-mc.com Organized by: boeld communication GmbH 2nd World Congress and Expo on Nanotechnology and Materials Science Date: 25th to 27th June 2018 Location: Dubai, United Arab Emirates Website: https://biocoreconferences.com/nanotechnology2018/index.php Contact person: Hany Organized by: Biocore 4th International Conference on Bio-based Polymers and Composites Date: 2nd to 6th September 2018 Location: Balatonfüred, Hungary Website: http://www.bipoco2018.hu Contact person: Dóra Tátraaljai Bio-based polymers and their blends, composites. Natural polymers and their modification. Natural fiber reinforced composites. Other raw materials based on natural resources. Biodegradation and environmental issues EMBO|EMBL Symposium: Organoids: Modelling Organ Development and Disease in 3D Culture Date: 10th to 13th September 2018 Location: Heidelberg, Germany Website: http://www.embo-embl-symposia.org/symposia/2018/EES18-08/index.html Contact person: events@embl.de<mailto:events@embl.de> Organized by: EMBL