Upscaled Skeletal Muscle Engineered Tissue with In Vivo Vascularization and Innervation Potential.

Engineering functional tissues of clinically relevant size (in mm-scale) in vitro is still a challenge in tissue engineering due to low oxygen diffusion and lack of vascularization. To address these limitations, a perfusion bioreactor was used to generate contractile engineered muscles of a 3 mm-thickness and a 8 mm-diameter. This study aimed to upscale the process to 50 mm in diameter by combining murine skeletal myoblasts (SkMbs) with human adipose-derived stromal vascular fraction (SVF) cells, providing high neuro-vascular potential in vivo. SkMbs were cultured on a type-I-collagen scaffold with (co-culture) or without (monoculture) SVF. Large-scale muscle-like tissue showed an increase in the maturation index over time (49.18 ± 1.63% and 76.63 ± 1.22%, at 9 and 11 days, respectively) and a similar force of contraction in mono- (43.4 ± 2.28 µN) or co-cultured (47.6 ± 4.7 µN) tissues. Four weeks after implantation in subcutaneous pockets of nude rats, the vessel length density within the constructs was significantly higher in SVF co-cultured tissues (5.03 ± 0.29 mm/mm2) compared to monocultured tissues (3.68 ± 0.32 mm/mm2) (p < 0.005). Although no mature neuromuscular junctions were present, nerve-like structures were predominantly observed in the engineered tissues co-cultured with SVF cells. This study demonstrates that SVF cells can support both in vivo vascularization and innervation of contractile muscle-like tissues, making significant progress towards clinical translation.

Bizonal cardiac engineered tissues with differential maturation features in a mid-throughput multimodal bioreactor.

Functional three-dimensional (3D) engineered cardiac tissue (ECT) models are essential for effective drug screening and biological studies. Application of physiological cues mimicking those typical of the native myocardium is known to promote the cardiac maturation and functionality in vitro. Commercially available bioreactors can apply one physical force type at a time and often in a restricted loading range. To overcome these limitations, a millimetric-scale microscope-integrated bioreactor was developed to deliver multiple biophysical stimuli to ECTs. In this study, we showed that the single application of auxotonic loading (passive) generated a bizonal ECT with a unique cardiac maturation pattern. Throughout the statically cultured constructs and in the ECT region exposed to high passive loading, cardiomyocytes predominantly displayed a round morphology and poor contractility ability. The ECT region with a low passive mechanical stimulation instead showed both rat- and human-origin cardiac cell maturation and organization, as well as increased ECT functionality.

A dynamic microscale mid-throughput fibrosis model to investigate the effects of different ratios of cardiomyocytes and fibroblasts.

Cardiac fibrosis is a maladaptive remodeling of the myocardium hallmarked by contraction impairment and excessive extracellular matrix deposition (ECM). The disease progression, nevertheless, remains poorly understood and present treatments are not capable of controlling the scarring process. This is partly due to the absence of physiologically relevant, easily operable, and low-cost in vitro models, which are of the utmost importance to uncover pathological mechanisms and highlight possible targets for anti-fibrotic therapies. In classic models, fibrotic features are usually obtained using substrates with scar mimicking stiffness and/or supplementation of morphogens such as transforming growth factor ß1 (TGF-ß1). Qualities such as the interplay between activated fibroblasts (FBs) and cardiomyocytes (CMs), or the mechanically active, three-dimensional (3D) environment, are, however, neglected or obtained at the expense of the number of experimental replicates achievable. To overcome these shortcomings, we engineered a micro-physiological system (MPS) where multiple 3D cardiac micro-tissues can be subjected to cyclical stretching simultaneously. Up to six different biologically independent samples are incorporated in a single device, increasing the experimental throughput and paving the way for higher yielding drug screening campaigns. The newly developed MPS was used to co-culture different ratios of neonatal rat CMs and FBs, investigating the role of CMs in the modulation of fibrosis traits, without the addition of morphogens, and in soft substrates. The expression of contractile stress fibers and of degradative enzymes, as well as the deposition of fibronectin and type I collagen were superior in microtissues with a low amount of CMs. Moreover, high CM-based microconstructs simulating a ratio similar to that of healthy tissues, even if subjected to both cyclic stretch and TGF-ß1, did not show any of the investigated fibrotic signs, indicating a CM fibrosis modulating effect. Overall, this in vitro fibrosis model could help to uncover new pathological aspects studying, with mid-throughput and in a mechanically active, physiologically relevant environment, the crosstalk between the most abundant cell types involved in fibrosis.

Impact on Mechanical Properties of 10 versus 20 Minute Treatment of Human Pericardium with Glutaraldehyde in OZAKI Procedure.

PURPOSE: The aim of this study was to analyze the effects of 10-minute (standard term) versus 20-minute treatment with glutaraldehyde (GA) on mechanical stability and physical strength of human pericardium in the setting of the OZAKI procedure. METHODS: Leftover pericardium (6 patients) was bisected directly after the operation, and one-half was further fixed for 10 additional minutes. Uniaxial tensile tests were performed and ultimate tensile strength (UTS), ultimate tensile strain (uts), and collagen elastic modulus were evaluated. RESULTS: Both treatments resulted in similar values of uniaxial stretching-generated elongations at rupture (10 minutes 25 ± 7 % vs. 20 minutes: 22 ± 5 %; p = 0.05), UTS (5.16 ± 2 MPa vs. 6.54 ± 3 MPa; p = 0.59), and collagen fiber stiffness (elastic modulus: 31.80 ± 15.05 MPa vs. 37.35 ± 15.78 MPa; p = 0.25). CONCLUSION: Prolongation of the fixation time of autologous pericardium has no significant effect on its mechanical stability; thus, extending the intraoperative treatment cannot be recommended.

Bioreactor Platform for Biomimetic Culture and in situ Monitoring of the Mechanical Response of in vitro Engineered Models of Cardiac Tissue.

In the past two decades, relevant advances have been made in the generation of engineered cardiac constructs to be used as functional in vitro models for cardiac research or drug testing, and with the ultimate but still challenging goal of repairing the damaged myocardium. To support cardiac tissue generation and maturation in vitro, the application of biomimetic physical stimuli within dedicated bioreactors is crucial. In particular, cardiac-like mechanical stimulation has been demonstrated to promote development and maturation of cardiac tissue models. Here, we developed an automated bioreactor platform for tunable cyclic stretch and in situ monitoring of the mechanical response of in vitro engineered cardiac tissues. To demonstrate the bioreactor platform performance and to investigate the effects of cyclic stretch on construct maturation and contractility, we developed 3D annular cardiac tissue models based on neonatal rat cardiac cells embedded in fibrin hydrogel. The constructs were statically pre-cultured for 5 days and then exposed to 4 days of uniaxial cyclic stretch (sinusoidal waveform, 10% strain, 1 Hz) within the bioreactor. Explanatory biological tests showed that cyclic stretch promoted cardiomyocyte alignment, maintenance, and maturation, with enhanced expression of typical mature cardiac markers compared to static controls. Moreover, in situ monitoring showed increasing passive force of the constructs along the dynamic culture. Finally, only the stretched constructs were responsive to external electrical pacing with synchronous and regular contractile activity, further confirming that cyclic stretching was instrumental for their functional maturation. This study shows that the proposed bioreactor platform is a reliable device for cyclic stretch culture and in situ monitoring of the passive mechanical response of the cultured constructs. The innovative feature of acquiring passive force measurements in situ and along the culture allows monitoring the construct maturation trend without interrupting the culture, making the proposed device a powerful tool for in vitro investigation and ultimately production of functional engineered cardiac constructs.

Fatty acid-based monolayer culture to promote in vitro neonatal rat cardiomyocyte maturation.

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.

Magnetic nanocomposite hydrogels and static magnetic field stimulate the osteoblastic and vasculogenic profile of adipose-derived cells.

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.

Modeling methodology for defining a priori the hydrodynamics of a dynamic suspension bioreactor. Application to human induced pluripotent stem cell culture.

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.

Paracrine potential of adipose stromal vascular fraction cells to recover hypoxia-induced loss of cardiomyocyte function.

Cell-based therapies show promising results in cardiac function recovery mostly through paracrine-mediated processes (as angiogenesis) in chronic ischemia. In this study, we aim to develop a 2D (two-dimensional) in vitro cardiac hypoxia model mimicking severe cardiac ischemia to specifically investigate the prosurvival paracrine effects of adipose tissue-derived stromal vascular fraction (SVF) cell secretome released upon three-dimensional (3D) culture. For the 2D-cardiac hypoxia model, neonatal rat cardiomyocytes (CM) were cultured for 5 days at < 1% (approaching anoxia) oxygen (O2 ) tension. Typical cardiac differentiation hallmarks and contractile ability were used to assess both the cardiomyocyte loss of functionality upon anoxia exposure and its possible recovery following the 5-day-treatment with SVF-conditioned media (collected following 6-day-perfusion-based culture on collagen scaffolds in either normoxia or approaching anoxia). The culture at < 1% O 2 for 5 days mimicked the reversible condition of hibernating myocardium with still living and poorly contractile CM (reversible state). Only SVF-medium conditioned in normoxia expressing a high level of the prosurvival hepatocyte-growth factor (HGF) and insulin-like growth factor (IGF) allowed the partial recovery of the functionality of damaged CM. The secretome generated by SVF-engineered tissues showed a high paracrine potential to rescue the nonfunctional CM, therefore resulting in a promising patch-based treatment of specific low-perfused areas after myocardial infarction.

A three-dimensional in vitro dynamic micro-tissue model of cardiac scar formation.

In vitro cardiac models able to mimic the fibrotic process are paramount to develop an effective anti-fibrosis therapy that can regulate fibroblast behaviour upon myocardial injury. In previously developed in vitro models, typical fibrosis features were induced by using scar-like stiffness substrates and/or potent morphogen supplementation in monolayer cultures. In our model, we aimed to mimic in vitro a fibrosis-like environment by applying cyclic stretching of cardiac fibroblasts embedded in three-dimensional fibrin-hydrogels alone. Using a microfluidic device capable of delivering controlled cyclic mechanical stretching (10% strain at 1 Hz), some of the main fibrosis hallmarks were successfully reproduced in 7 days. Cyclic strain indeed increased cell proliferation, extracellular matrix (ECM) deposition (e.g. type-I-collagen, fibronectin) and its stiffness, forming a scar-like tissue with superior quality compared to the supplementation of TGFß1 alone. Taken together, the observed findings resemble some of the key steps in the formation of a scar: (i) early fibroblast proliferation, (ii) later phenotype switch into myofibroblasts, (iii) ECM deposition and (iv) stiffening. This in vitro scar-on-a-chip model represents a big step forward to investigate the early mechanisms possibly leading later to fibrosis without any possible confounding supplementation of exogenous potent morphogens.

Sensitivity of human pluripotent stem cells to insulin precipitation induced by peristaltic pump-based medium circulation: considerations on process development.

Controlled large-scale production of human pluripotent stem cells (hPSCs) is indispensable for their envisioned clinical translation. Aiming at advanced process development in suspension culture, the sensitivity of hPSC media to continuous peristaltic pump-based circulation, a well-established technology extensively used in hydraulically-driven bioreactors, was investigated. Unexpectedly, conditioning of low protein media (i.e. E8 and TeSR-E8) in a peristaltic pump circuit induced severe viability loss of hPSCs cultured as aggregates in suspension. Optical, biochemical, and cytological analyses of the media revealed that the applied circulation mode resulted in the reduction of the growth hormone insulin by precipitation of micro-sized particles. Notably, in contrast to insulin depletion, individual withdrawal of other medium protein components (i.e. bFGF, TGFß1 or transferrin) provoked minor reduction of hPSC viability, if any. Supplementation of the surfactant glycerol or the use of the insulin analogue Aspart did not overcome the issue of insulin precipitation. In contrast, the presence of bovine or human serum albumin (BSA or HSA, respectively) stabilized insulin rescuing its content, possibly by acting as molecular chaperone-like protein, ultimately supporting hPSC maintenance. This study highlights the potential and the requirement of media optimization for automated hPSC processing and has broad implications on media development and bioreactor-based technologies.

A Versatile Bioreactor for Dynamic Suspension Cell Culture. Application to the Culture of Cancer Cell Spheroids.

A versatile bioreactor suitable for dynamic suspension cell culture under tunable shear stress conditions has been developed and preliminarily tested culturing cancer cell spheroids. By adopting simple technological solutions and avoiding rotating components, the bioreactor exploits the laminar hydrodynamics establishing within the culture chamber enabling dynamic cell suspension in an environment favourable to mass transport, under a wide range of tunable shear stress conditions. The design phase of the device has been supported by multiphysics modelling and has provided a comprehensive analysis of the operating principles of the bioreactor. Moreover, an explanatory example is herein presented with multiphysics simulations used to set the proper bioreactor operating conditions for preliminary in vitro biological tests on a human lung carcinoma cell line. The biological results demonstrate that the ultralow shear dynamic suspension provided by the device is beneficial for culturing cancer cell spheroids. In comparison to the static suspension control, dynamic cell suspension preserves morphological features, promotes intercellular connection, increases spheroid size (2.4-fold increase) and number of cycling cells (1.58-fold increase), and reduces double strand DNA damage (1.5-fold reduction). It is envisioned that the versatility of this bioreactor could allow investigation and expansion of different cell types in the future.

Comparison of symmetric hemodialysis catheters using computational fluid dynamics.

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.