Department of Food Processing Technology and Management
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Item TOWARDS MIMICKING THE FETAL LIVER NICHE: THE INFLUENCE OF ELASTICITY AND OXYGEN TENSION ON HEMATOPOIETIC STEM/PROGENITOR CELLS CULTURED IN 3D FIBRIN HYDROGELS(MDPI, 2020-09-02) Christian, Garcia-Abrego; Samantha, Zaunz; Burak, Toprakhisar; Ramesh, Subramani; Olivier, Deschaume; Stijn, Jooken; Manmohan, Bajaj; Herman, Ramon; Catherine, Verfaillie; Carmen, Bartic; Jennifer, PattersonHematopoietic stem/progenitor cells (HSPCs) are responsible for the generation of blood cells throughout life. It is believed that, in addition to soluble cytokines and niche cells, biophysical cues like elasticity and oxygen tension are responsible for the orchestration of stem cell fate. Although several studies have examined the effects of bone marrow (BM) niche elasticity on HSPC behavior, no study has yet investigated the effects of the elasticity of other niche sites like the fetal liver (FL), where HSPCs expand more extensively. In this study, we evaluated the effect of matrix stiffness values similar to those of the FL on BM-derived HSPC expansion. We first characterized the elastic modulus of murine FL tissue at embryonic day E14.5. Fibrin hydrogels with similar stiffness values as the FL (soft hydrogels) were compared with stiffer fibrin hydrogels (hard hydrogels) and with suspension culture. We evaluated the expansion of total nucleated cells (TNCs), Lin−/cKit+cells, HSPCs (Lin−/Sca+/cKit+ (LSK) cells), and hematopoietic stem cells (HSCs: LSK- Signaling Lymphocyte Activated Molecule (LSK-SLAM) cells) when cultured in 5% O2 (hypoxia) or in normoxia. After 10 days, there was a significant expansion of TNCs and LSK cells in all culture conditions at both levels of oxygen tension. LSK cells expanded more in suspension culture than in both fibrin hydrogels, whereas TNCs expanded more in suspension culture and in soft hydrogels than in hard hydrogels, particularly in normoxia. The number of LSK-SLAM cells was maintained in suspension culture and in the soft hydrogels but not in the hard hydrogels. Our results indicate that both suspension culture and fibrin hydrogels allow for the expansion of HSPCs and more differentiated progeny whereas stiff environments may compromise LSK-SLAM cell expansion. This suggests that further research using softer hydrogels with stiffness values closer to the FL niche is warranted.Item THE INFLUENCE OF SWELLING ON LOCAL ELASTIC PROPERTIES OF POLYACRYLAMIDE HYDROGELS(Open Science Framework, 2017) Ramesh, Subramani; Alicia, Izquierdo-Alvarez; Pinaki, Bhattacharya; Mathieu, Meerts; Paula, Moldenaers; Herman, Ramon; Hans Van, OosterwyckPolyacrylamide (PAM)hydrogelsarecommonly usedas soft cell culture substrates for cell mechanical and mechanobiological studiesbecause oftheir tunable stiffness,easeof handling, transparent natureand surface functionalization to promote cell adhesion. The dependence of bulk rheological and local elastic properties (for example, as assessed by means of Atomic Force Microscopyor AFM) of PAM hydrogels onmonomer and cross-linkerconcentrationsand on polymerization temperature havebeen extensively investigated.PAM hydrogels are known to swell, which may affect their elastic properties and therefore may complicate the interpretation ofcell culturingexperiments because of a lack of control of substrate stiffness. Direct measurements of the effect of swelling on PAM elastic properties are scarce. We report here, for the first time, the direct observation of swelling (by measuring the volumetric swelling ratio) and its influence onlocal elastic properties, as measuredby AFM. Bulk rheological measurements were performed as well to enable the comparison between local and global elastic properties during and after hydrogel polymerization. Four PAM hydrogel compositions were considered,with corresponding storage shear moduliof 4530 Pa(termed stiffest), 2900 Pa(stiff), 538 Pa(soft)and 260 Pa(softest) as measured immediately after polymerization. These values as well as values obtained during hydrogel polymerization were found to be in good agreement with the local elastic moduli derived from AFM and assuming hydrogel incompressibility. After polymerization, the hydrogels were subjected to swelling conditions over six days in phosphate buffered salineand swelling ratios and local elastic moduli were measured each day.Additional short term measurements (at 0, 3, 6, 9, 12 and 24 hours) were performed for the soft and softest gelsto study their swelling kinetics in more detail. Swelling ratio and local elastic modulus did not change with time for the stiffest and stiff gels, while for the soft and softest gels substantialchanges between Day 0 and Day 1were found for both swelling ratio (21.6%and 133% increase for soft and softest gel respectively) and local elastic modulus (33.7%and 33.3% decrease for soft and softest gel respectively). Experimental data werefurther analysed theoretically by combining models of ideal elastomeric gels with a poroelastic swelling kinetics model, which confirmed the validity of observed trends with respect to literature data. The results demonstrate that swelling can have an important effect on PAM elastic properties and must be taken into account when using PAM as a cell culture substrate, particularly for PAM gels with low monomer and cross-linker concentrationsItem IMMERSED BOUNDARY MODELS FOR QUANTIFYING FLOW-INDUCED MECHANICAL STIMULI ON STEM CELLS SEEDED ON 3D SCAFFOLDS IN PERFUSION BIOREACTORS(PLOS Computational Biology, 2016-09-22) Yann, Guyot; Bart, Smeets; Tim, Odenthal; Ramesh, Subramani; Frank P, Luyten; Herman, Ramon; Ioannis, Papantoniou; Liesbet, GerisPerfusion bioreactors regulate flow conditions in order to provide cells with oxygen, nutrients and flow-associated mechanical stimuli. Locally, these flow conditions can vary depending on the scaffold geometry, cellular confluency and amount of extra cellular matrix deposition. In this study, a novel application of the immersed boundary method was introduced in order to represent a detailed deformable cell attached to a 3D scaffold inside a perfusion bioreactor and exposed to microscopic flow. The immersed boundary model permits the prediction of mechanical effects of the local flow conditions on the cell. Incorporating stiffness values measured with atomic force microscopy and micro-flow boundary conditions obtained from computational fluid dynamics simulations on the entire scaffold, we compared cell deformation, cortical tension, normal and shear pressure between different cell shapes and locations. We observed a large effect of the precise cell location on the local shear stress and we predicted flow-induced cortical tensions in the order of 5 pN/μm, at the lower end of the range reported in literature. The proposed method provides an interesting tool to study perfusion bioreactors processes down to the level of the individual cell’s micro-environment, which can further aid in the achievement of robust bioprocess control for regenerative medicine applications.Item ROBUST SCALABLE SYNTHESIS OF A BIS-UREA DERIVATIVE FORMING THIXOTROPIC AND CYTOCOMPATIBLE SUPRAMOLECULAR HYDROGELS(Royal Society of Chemistry, 2019-06-06) Laurens A J, Rutgeerts; Al, Halifa Soulta; Ramesh, Subramani; Burak, Toprakhisar; Herman, Ramon; Monissa C, Paderes; Wim M, De Borggraeve; Jennifer, PattersonSynthetic hydrogels address a need for affordable, industrially scalable scaffolds for tissue engineering. Herein, a novel low molecular weight gelator is reported that forms self-healing supramolecular hydrogels. Its robust synthesis can be performed in a solvent-free manner using ball milling. Strikingly, encapsulated cells spread and proliferate without specific cell adhesion ligands in the nanofibrous material.Item IMMERSED BOUNDARY MODELSFOR QUANTIFYING FLOW-INDUCED MECHANICAL STIMULI ON STEM CELLS SEEDED ON 3D SCAFFOLDS IN PERFUSION BIOREACTORS(PLOS Computational Biology, 2016-09-22) Yann, Guyot; Bart, Smeets; Tim, Odentha; Ramesh, Subramani; Frank P, Luyten; Herman, Ramon; Ioannis, Papantoniou; Liesbet, GerisPerfusion bioreactors regulate flow conditions in order to provide cells with oxygen, nutrients and flow-associated mechanical stimuli. Locally, these flow conditions can vary depending on the scaffold geometry, cellular confluency and amount of extra cellular matrix deposition. In this study, a novel application of the immersed boundary method was introduced in order to represent a detailed deformable cell attached to a 3D scaffold inside a perfusion bioreactor and exposed to microscopic flow. The immersed boundary model permits the prediction of mechanical effects of the local flow conditions on the cell. Incorporating stiffness values measured with atomic force microscopy and micro-flow boundary conditions obtained from computational fluid dynamics simulations on the entire scaffold, we compared cell deformation, cortical tension, normal and shear pressure between different cell shapes and locations. We observed a large effect of the precise cell location on the local shear stress and we predicted flow-induced cortical tensions in the order of 5 pN/μm, at the lower end of the range reportedin literature. The proposed method provides an interesting tool to study perfusion bioreactors processes down to the level of the individual cell’s micro-environment, which can furtheraid in the achievement of robust bioprocess control for regenerative medicine applications.Item THE INFLUENCE OF SWELLING ON ELASTIC PROPERTIES OF POLYACRYLAMIDE HYDROGELS(Frontiers in Materials, 2020-07-23) Ramesh, Subramani; Alicia, Izquierdo-Alvarez; Pinaki, Bhattacharya; Mathieu, Meerts; Paula, Moldenaers; Herman, Ramon; Hans, Van OosterwyckPolyacrylamide (PAM) hydrogels are commonly used as substrates for cell mechanical and mechanobiological studies because of their tunable stiffness and ease of handling. The dependence of bulk rheological and local elastic properties (assessed by Atomic Force Microscopy, or AFM) of PAM hydrogels on its composition and polymerization temperature has been extensively studied. PAM hydrogels swell when immersed in media, but the influence of swelling on local elastic properties is poorly characterized. Direct measurements of the effect of swelling on PAM elastic properties are scarce. We report here, for the first time, the direct measurements of volumetric swelling and local elastic properties of PAM gels throughout the post-polymerization swelling process until equilibrium. First, local and global elastic properties (measured by rheology), were obtained during polymerization in the absence of swelling, and showed good agreement with each other. Four PAM hydrogel compositions were characterized thus, with corresponding storage shear moduli (as measured immediately after polymerization) of 4,530 Pa (termed stiffest), 2,900 Pa (stiff), 538 Pa (soft), and 260 Pa (softest). Next, all compositions were subjected to swelling in phosphate buffered saline. Swelling ratios and local elastic moduli were measured at 0, 3, 6, 9, 12, and 24 h post-polymerization for the soft and softest compositions, and once daily till 6 days post-polymerization for all four compositions. For the stiffest and stiff gels, swelling ratio, and local elastic modulus changed negligibly with time, while for the soft and softest gels, substantial changes between Day 0 and Day 1 were found for both swelling ratio (increased by 21.6 and 133%, respectively), and local elastic modulus decreased (by 33.7 and 33.3%, respectively), substantially. Experimental data were analyzed by a model that combined ideal elastomer mechanics and poroelastic swelling kinetics model. Model predictions confirmed the validity of present measurements with respect to past studies where swelling and elastic properties were not measured simultaneously. The present study underlines the important effect swelling can have on PAM elastic properties and provides detailed quantitative data to guide the duration taken to reach equilibrium—a useful information for cell mechanics experiments. In addition, the simultaneous measurements of swelling and local elastic moduli provide novel data for the validation of theoretical models.Item TOWARDS MIMICKING THE FETAL LIVER NICHE: THE INFLUENCE OF ELASTICITY AND OXYGEN TENSION ON HEMATOPOIETIC STEM/PROGENITOR CELLS CULTURED IN 3D FIBRIN HYDROGELS(2020-09-02) Christian, Garcia-Abrego; Samantha, Zaunz; Burak, Toprakhisar; Ramesh, Subramani; Olivier, Deschaume; Stijn, Jooken; Manmohan, Baja; Herman, Ramon; Catherine, Verfaillie; Carmen, Bartic; Jennifer, PattersonHematopoietic stem/progenitor cells (HSPCs) are responsible for the generation of blood cells throughout life. It is believed that, in addition to soluble cytokines and niche cells, biophysical cues like elasticity and oxygen tension are responsible for the orchestration of stem cell fate. Although several studies have examined the effects of bone marrow (BM) niche elasticity on HSPC behavior, no study has yet investigated the effects of the elasticity of other niche sites like the fetal liver (FL), where HSPCs expand more extensively. In this study, we evaluated the effect of matrix stiffness values similar to those of the FL on BM-derived HSPC expansion. We first characterized the elastic modulus of murine FL tissue at embryonic day E14.5. Fibrin hydrogels with similar stiffness values as the FL (soft hydrogels) were compared with stiffer fibrin hydrogels (hard hydrogels) and with suspension culture. We evaluated the expansion of total nucleated cells (TNCs), Lin−/cKit+ cells, HSPCs (Lin−/Sca+/cKit+ (LSK) cells), and hematopoietic stem cells (HSCs: LSK- Signaling Lymphocyte Activated Molecule (LSK-SLAM) cells) when cultured in 5% O2 (hypoxia) or in normoxia. After 10 days, there was a significant expansion of TNCs and LSK cells in all culture conditions at both levels of oxygen tension. LSK cells expanded more in suspension culture than in both fibrin hydrogels, whereas TNCs expanded more in suspension culture and in soft hydrogels than in hard hydrogels, particularly in normoxia. The number of LSK-SLAM cells was maintained in suspension culture and in the soft hydrogels but not in the hard hydrogels. Our results indicate that both suspension culture and fibrin hydrogels allow for the expansion of HSPCs and more differentiated progeny whereas stiff environments may compromise LSK-SLAM cell expansion. This suggests that further research using softer hydrogels with stiffness values closer to the FL niche is warranted.