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Three-dimensional dynamic suspension is becoming an effective cell culture method for a wide range of bioprocesses, with an increasing number of bioreactors proposed for this purpose. The complex hydrodynamics establishing within these devices affects bioprocess outcomes and efficiency, and usually expensive in vitro trial-and-error experiments are needed to properly set the working parameters. Here we propose a methodology to define a priori the hydrodynamic working parameters of a dynamic suspension bioreactor, selected as a test case because of the complex hydrodynamics characterizing its operating condition. A combination of computational and analytical approaches was applied to generate operational guideline graphs for defining a priori specific working parameters. In detail, 43 simulations were performed under pulsed flow regime to characterize advective transport within the device depending on different operative conditions, i.e., culture medium flow rate and its duty cycle, cultured particle diameter, and initial particle suspension volume. The operational guideline graphs were then used to set specific hydrodynamic working parameters for an in vitro proof-of-principle test, where human induced pluripotent stem cell (hiPSC) aggregates were cultured for 24 h within the bioreactor. The in vitro findings showed that, under the selected pulsed flow regime, sedimentation was avoided, hiPSC aggregate circularity and viability were preserved, and culture heterogeneity was reduced, thus confirming the appropriateness of the a priori method. This methodology has the potential to be adaptable to other dynamic suspension devices to support experimental studies by providing in silico-based a priori knowledge, useful to limit costs and to optimize culture bioprocesses.

The development of functional and reliable in vitro cardiac models composed of fully mature cardiomyocytes is essential for improving drug screening test quality, therefore, the success of clinical trial outcomes. In their lifespan, cardiomyocytes undergo a dynamic maturation process from the fetal to adult stage, radically changing their metabolism, morphology, contractility and electrical properties. Before employing cells of human origin, in vitro models often use neonatal rat cardiomyocytes (NRCM) to obtain key proof-of-principles. Nevertheless, NRCM monolayers are prone to de-differentiate when maintained in culture. Supplementation of free fatty acids (FFA), the main energy source for mature cardiomyocytes, and co-culture with fibroblasts are each by itself known to promote the shift from fetal to adult cardiomyocytes. Using a co-culture system, our study investigates the effects of FFA on the cardiomyocyte phenotype in comparison to glucose as typical fetal energy source, and to 10% serum used as standard control condition. NRCM decreased their differentiation status and fibroblasts increased in number after 7days of culture in the control condition. On the contrary, both glucose- and FFA-supplementation better preserved protein expression of myosin-light-chain-2v, a marker of mature cardiomyocytes, and the fibroblast number at levels similar to those found in freshly isolated NRCM. Nevertheless, compared to glucose, FFA resulted in a significant increase in sarcomere striation and organization. Our findings constitute an important step forward towards the definition of the optimal culture conditions, highlighting the possible benefits of a further supplementation of specific FFA to promote CM maturation in a co-culture system with FB.

Exposure of cells to externally applied magnetic fields or to scaffolding materials with intrinsic magnetic properties (magnetic actuation) can regulate several biological responses. Here, we generated novel magnetized nanocomposite hydrogels by incorporation of magnetic nanoparticles (MNPs) into polyethylene glycol (PEG)-based hydrogels containing cells from the stromal vascular fraction (SVF) of human adipose tissue. We then investigated the effects of an external Static Magnetic Field (SMF) on the stimulation of osteoblastic and vasculogenic properties of the constructs, with MNPs or SMF alone used as controls. MNPs migrated freely through and out of the material following the magnetic gradient. Magnetically actuated cells displayed increased metabolic activity. After 1 week, the enzymatic activity of Alkaline Phosphatase (ALP), the expression of osteogenic markers (Runx2, Collagen I, Osterix), and the mineralized matrix deposition were all augmented as compared to controls. With magnetic actuation, strong activation of endothelial, pericytic and perivascular genes paralleled increased levels of VEGF and an enrichment in the CD31+ cells population. The stimulation of signaling pathways involved in the mechanotransduction, like MAPK8 or Erk, at gene and protein levels suggested an effect mediated through the mechanical stimulation. Upon subcutaneous implantation in mice, magnetically actuated constructs exhibited denser, more mineralized and faster vascularized tissues, as revealed by histological and micro-computed tomographic analyses. The present study suggests that magnetic actuation can stimulate both the osteoblastic and vasculogenic potentials of engineered bone tissue grafts, likely at least partially by mechanically stimulating the function of progenitor cells.

Purpose Symmetric-tip dialysis catheters have become alternative devices because of low access recirculation and ease of tip positioning. Flow characteristics of three symmetric catheters were compared based on computational fluid dynamics (CFD) as they relate to catheter function. Materials And Methods In Palindrome, GlidePath, and VectorFlow catheters, a computational fluid dynamics–based approach was used to assess (i) regions of flow separation, which are prone to thrombus development; (ii) shear-induced platelet activation potency; (iii) recirculation; and (iv) venous outflow deflection. A steady-state, laminar flow model simulated catheter tip position within the superior vena cava. Catheter performance was investigated at high hemodialysis flow rate (400 mL/min). Blood was assumed as a Newtonian fluid. Results Wide regions of flow separation downstream of the Palindrome side slot and close to the distal tip were observed in forward and reversed line configurations. Geometric asymmetry of the distal guide wire aperture of the GlidePath catheter produced the highest levels of inverted velocity flow when run in reversed configuration. The lowest mean shear-induced platelet activation was exhibited by GlidePath and VectorFlow catheters; the Palindrome catheter exhibited 152% higher overall platelet activation potency. All catheters were associated with a recirculation close to zero; the helically contoured lumens of the VectorFlow catheter produced the greatest amount of deflection of venous flow away from the arterial lumen. Conclusions The VectorFlow catheter produced less shear-induced platelet activation than the Palindrome catheter and less flow separation than the Palindrome and GlidePath catheters irrespective of line configuration. These findings have potential implications for differences in thrombogenic risk during clinical performance of these catheters.