1. Discussion and Conclusions Proliferation of MSCs in 3D vs. 2D Culture Microgel cell carrier preparation Revital Goldshmid and Dror Seliktar Faculty of Biomedical Engineering, Technion-The Israel Institute of Technology, Haifa, Israel ABSTRACT Human mesenchymal stem cells (hMSC) are of significant interest as a renewable source of therapeutically useful cells. Even as scientists uncover precisely how to manipulate hMSC cultures toward directed pathways of differentiation or self-renewal, their routine commercial and clinical applications will still require cultivation and bioprocessing efficiency that cannot be provided by conventional adherent cell culture technologies. The development of scalable methodologies for handling hMSCs necessitates an approach premised on the use of three dimensional (3-D) suspension cultures that are readily adaptable to large-scale bioreactors. In this context, hydrogel biomaterials may offer the versatility required to undertake the difficult task of developing 3-D bioprocessing technologies for stem cells that can be applied in commercial clinical settings. We focus on developing a methodology for encapsulating hMSCs in hydrogel biomaterials to be used as a routine, efficient, and scalable solution for hMSC bioprocessing. The biomaterial technology is premised on a semi-synthetic hydrogel made by conjugating fibrinogen with polyethylene glycol diacrylate (PF), developed in our laboratory for tissue engineering, and now adapted for micro-carriers suitable for suspension culture bioreactors. 3D milieu show an advantage to MSCs proliferation in the long term compared to a 2D plate culture. In addition, alteration of storage modulus (G’) of the PEG-Fib hydrogel, enables us to control cells differentiation and proliferation: Neurogenesis, Myogenesis and Chondrogenesis of MSCs is observed respective to increasing storage moduli. Thus, the interplay between physical and biochemical signals present in the stem cells milieu guide cells fate determination. MSCs in 3D microgels cultured for long periods (up to 21 days) present an increase in proliferation, unlike the decrease of S/G2/M phase rates in 2D plates. Those results are correlative with cells quantity change with time. A significant difference of myogenesis markers expression is observed in 3D MSCs culture compared to 2D plates culture along with satellite cell morphology; 2D plates culture show flat cells less differentiate morphology than 3D microgels. Chondrogenesis shows better differentiation kinetics in 3D compared to 2D culture. Ongoing neurogenesis is observed along 21 days of experiment, exhibiting a preferential differentiation towards neurons rather then Glia cells. Myogenesis of MSCs in 3D vs. 2D Culture 3D Microgels show an advantage in long term culture, presenting an accelerated proliferation, as observed by the high S/G2/M phase rates. The cell quantity is significantly higher in 3D microgels then in 2D plates as shown by the better cell/area rate. MSCs cultured in 3D Microgels shows elongation and proliferation morphology. References Seliktar D., Designing cell-compatible hydrogels for biomedical applications. Science. 336(6085):1124-8, 2012. 24. Appelman, T., Mizrahi, J., Elisseeff, J., Seliktar, D., “The differential effect of scaffold composition and architecture on chondrocytes response to mechanical stimulation, Biomaterials, 30(4): 518-25, 2009. Junmin Lee, et al, Directing stem cell fate on hydrogel substrates by controlling cell geometry, matrix mechanics and adhesion ligand composition, Biomaterials 34 (2013) Andrew S. Rowlands et al, Directing osteogenic and myogenic differentiation of MSCs: interplay of stiffness and adhesive ligand presentation, Am J Physiol Cell Physiol 295:C1037-C1044, 2008 Magne D, Vinatier C, Julien M, Weiss P, Guicheux J. Mesenchymal stem cell therapy to rebuild cartilage. Trends Mol Med 11: 519–526, 2005. Iwona Grabowska et al, Myogenic Potential of Mesenchymal Stem Cells - the Case of Adhesive Fraction of Human Umbilical Cord Blood Cells, Current Stem Cell Research & Therapy, 2013, 8, 82-90 Lab Website: http://brm.technion.ac.il Acknowledgments Surface coating with glass nano-particles in acetone Acetone evaporation leaves a superhydrophobic surface Hydrogel precursor with cells dripped onto surface Microgel crosslinking with photopolymerization (UV) Chondrogenesis of MSCs in 3D vs. 2D Culture Neurogenesis of MSCs in 3D microgels MSCs in 3D (mid stiffed PF) Microgels show accelerated smooth muscle actin (S.M.A, late myogenesis marker) expression indicating advanced cell differentiation stages. High S.M.A expression is observed in 3D MSCs culture on day 14 along with satellite cell morphology; 2D plates culture show flat cells morphology and less S.M.A expression. S.M.A-FITC, CD90-647, Actin-TriC, Nuclei-Dapi. Lite. Bar=40µm Satelite and Myofiber differentiated cells in 3D microgel (day14) differentiated cells on 2D plates (day 14) 3D Microgel 2D Plate Collagen-II expreesion of differentiated MSCs in 3D PEG-Fibrinogen microgels, day14. Aggrecan expreesion of differentiated MSCs in 3D PEG-Fibrinogen microgels, day14. The research leading to these results has received funding from the European Union FP7 Program under grant agreement number262948 (BIODESIGN) Cell Cycle Cell Quantity Smooth Muscle Cells Actin (S.M.A) expression Early chondrogenesis (Sox9) expression Neurogenic pre- and mature markers expression Microgels containing MSCs cultured in stirred bioreactor Detected by DNA stain (PI stain) on a flow cytometer Analyzed by IN Cell according to Actin and Nucleus markers Proliferating MSCs segmentation in 3D culture on day 3 Nuclei (Dapi), Actin (phalloidin TriC). Bar=10 μ m Cell Morphology MSCs cultured in 3D PEG-Fibrinogen (high stiffed) microgels for 14 days show better differentiation kinetics in 3D compared to 2D culture, as expressed by the early chondrogenic marker Sox9. After 14 days of culture, high levels of late chondrogenic markers (Collagen II and Agrecan) are observed in 3D cultures, observed on a significantly lower extent in 2D cultured MSCs. Coll-II-Cy5, Aggrecan-Cy3, CD90-647, Nuclei-Sytox green. Bar=40µm β -Tubulin-III and Olig2 expression of MSCs culture in 3D microgels on day 14 MSCs cultured in low stiffed PEG-Fibrinogen 3D microgels show increasing β-Tubulin-III (positive marker) and decreasing CD-105 (negative marker) expressions, indicating neurogenesis. Furthermore, increasing Vimentin (early naurogenesis marker) expression implies ongoing differentiation of MSCs into neurons in the course of 21 days. 3D microgels allow cells to differentiate to neurons (β-Tubulin-III, Olig2) more than to Glia cells (GFAP) and also improve neurogenesis compared to 2D plates or stiffed 3D microgels (data not shown). β-Tubulin-III-APC, Olig2-565, Nuclei-Dapi. Bar=10µm Proliferating MSCs on day 7 Actin (phalloidin TriC). Nuclei (Cytox-Green), Bar=25 μ m 2D plate 3D microgel