Hydrogel Microsphere Cell Carriers for Cell Expansion in Suspension Culture: Implications for Stem Cell Therapeutics Revital Goldshmid and Dror Seliktar Faculty of Biomedical Engineering, Technion-The Israel Institute of Technology, Haifa, Israel ABSTRACT The development of scalable methodologies for handling human stem cells (hSC) necessitates an approach premised on the use of three dimensional (3-D) suspension cultures that are readily adaptable to large-scale bioreactors. In this project, encapsulating hydrogel biomaterials were used for developing a 3D bioprocessing methodology for stem cells through a straightforward cell inoculation into microspheres, in situ self-renewal in the microsphere culture system and mild cell recovery into a solution phase. For creating the microsphere carriers, we employed a temperature-responsive semi-synthetic hydrogel material produced by the conjugation of poloxamers to fibrinogen. Mesenchymal cells were encapsulated and cultivated in the hydrogel microspheres using suspension bioreactors for up to 14 days. The viability, cell cycle and proliferation were characterized and indicated comparable expansion results to 2-D tissue cultures on petri dishes. Cell recovery from the hydrogels was accomplished enzymatically with high yields and minimal disruption to cell viability. We conclude that a biomaterial-based method can be successfully applied for suspension expansion of cells with controlled cell proliferation through cell-cell and cell-material interactions. Moreover, hydrogel microsphere cell carriers such as the ones described herein may offer a tangible solution to the growing demand for commercial-scale stem cell bioprocessing practices. FF-127 Hydrogel Preparation MSC Morphology in 3D vs. 2D Culture Day 1 Day 4 Day 8 Day 15 Day 20 Biological components Synthetic component Bovine Fibrinogen: MW = 166 kDa α, β, γ chains Pluronic F127 MW = 12.6 kDa Poly- acryl groups FF-127 2 RGD motifs Pluronic-Fib (FF-127) 2D Plate Pluronic®F127 is conjugated to fibrinogen (Fibrinogen- F127, FF-127). The morphology of MSCs cultured in FF-127 hydrogels and on 2D plates are shown. Cell staining: Phalloidin-TRITC (red) stained f-actin, SYTOX (green) stained nuclei. MSCs on 2D plates formed a uniform layer whereas MSCs in FF-127 exhibited distinct morphogenesis patterns. Scale bar=50μm Viability of MSCs in 3D vs. 2D Culture FF-127 2D Plate PI Viability test detected by FACS (MSCs): Viability: Quantitative propidium iodide (PI) viability assay; Qualitative live/dead staining with Calcein (Green) and Ethidium (Red). Results: MSCs survive on 2D plates as well as in FF-127 3D cultures (over 80% viability detected). Scale bar=100μm. Calcein/Ethidium (live/dead) assay (MSCs): Day 1 Day 4 Day 8 Day 15 Day 20 MSC Phenotype in 3D vs. 2D Culture MSCs in FF127 (3D) MSCs on culture dish (2D) CD31-PE (- cont) CD105-FITC (+ cont) FF-127 2D Plate Endoglin (CD105): an endothelial homodimeric membrane antigen is a marker to phenotypically characterize mesenchymal stem cells (MSCs) used as positive marker for MSCs. Platelet endothelial cell adhesion molecule (PECAM-1): is a well known negative marker for mesenchymal stem cells (MSCs), also know as CD31. Results: MSC phenotype is better retained in 3D culture than on 2D culture dishes. MSCs Proliferation in 3D vs. 2D Culture Proliferation assay: BRDU incorporated into newly synthesized DNA 20h before detection. Cells harvested and co-stained with FITC anti-BrdU fluorescent antibodies and 7AAD. Levels of cell-associated BrdU and 7AAD are then measured by flow cytometry (LSR-II) to determine proliferation and cell-cycle kinetics. Cell-Cycle Analysis of MSCs in 3D vs. 2D Culture Assay: determines the percent of MSCs in S- phase (synthesis phase) and G2/M (Mitosis and cytokinesis phase) as detected by DNA stain (PI stain) on a flow cytometer. Results: Early on, there are similar cell-cycle rate of MSCs in FF-127 compared to 2D culture dish. Later stages, more MSCs proliferating in FF-127 3D culture compared to 2D culture dish. BRDU levels detected by FACS (MSCs): FF-127 (3D) Culture dish (2D) Day 1 Day 4 Day 8 Day 15 Day 20 Discussion and Conclusions References MSCs exhibit high viability and proliferation in FF127 3D culture, and survived as good as on 2D tissue culture plates. MSCs phenotype, measured with CD105 and CD31 levels, was retained in FF127 3D culture as compared to 2D culture dishes. The MSCs in 3D formed morphology that was more similar to in vivo, when compared to 2D culture dish MSC morphology. The FF-127 3D cell culture hydrogel system is adaptive to suspension bioreactors, providing an ability to scale up MSCs expansion without the expense of costly 2D culture methodologies. Seliktar D., Designing cell-compatible hydrogels for biomedical applications. Science. 336(6085):1124-8, 2012. Shachaf Y, Gonen-Wadmany M, Seliktar D., The biocompatibility of PluronicF127 fibrinogen-based hydrogels. Biomaterials. 31(10):2836-47, 2010. Acknowledgments We acknowledge the financial support of the European Union FP7 Program, the Russell Berrie Nanotechnology Institute, and the Lorry I. Lokey Interdisciplinary Center for Life Sciences and Engineering. Lab Website: http://brm.technion.ac.il