Bruna Vinci

Criticality of the Biological and Physical Stimuli Array Inducing Resident Cardiac Stem Cell Determination

The replacement of injured cardiac contractile cells with stem cell‐derived functionally efficient cardiomyocytes has been envisaged as the resolutive treatment for degenerative heart diseases. Nevertheless, many technical issues concerning the optimal procedures to differentiate and engraft stem cells remain to be answered before heart cell therapy could be routinely used in clinical practice. So far, most studies have been focused on evaluating the differentiative potential of different growth factors without considering that only the synergistic cooperation of biochemical, topographic, chemical, and physical factors could induce stem cells to adopt the desired phenotype. The present study demonstrates that the differentiation of cardiac progenitor cells to cardiomyocytes does not occur when cells are challenged with soluble growth factors alone, but requires strictly controlled procedures for the isolation of a progenitor cell population and the artifactual recreation of a microenvironment critically featured by a fine‐tuned combination of specific biological and physical factors. Indeed, the scaffold geometry and stiffness are crucial in enhancing growth factor differentiative effects on progenitor cells. The exploitation of this concept could be essential in setting up suitable procedures to fabricate functionally efficient engineered tissues.

Modular bioreactor for primary human hepatocyte culture: Medium flow stimulates expression and activity of detoxification genes

Down‐regulation of detoxification genes, notably cytochrome P450 (CYPs), in primary hepatocyte cultures is a long‐standing and major concern. We evaluated the influence of medium flow in this model. Hepatocytes isolated from 12 different liver donors were cultured either in a multichamber modular bioreactor (MCmB, flow rate 250–500 μL/min) or under standard/static conditions, and the expression of 32 genes, enzyme activities and biological parameters were measured 7–21 days later. mRNA expression of genes involved in xenobiotic/drug metabolism and transport, including CYP1A1, 1A2, 2B6, 2C9, 3A4 (and activities for some of them), UDP‐glucuronosyltransferase (UGT) 1A1, UGT2B4, UGT2B7, glutathione S‐transferase (GSTα), and multidrug resistance protein 1 (MDR1) and MRP2, were specifically up‐regulated by medium flow as compared with static controls in all cultures tested. In 2‐week‐old cultures, expression of detoxification genes reached levels close to or higher than those measured in freshly isolated hepatocytes. In contrast, CYP2D6 and most of other tested genes were not affected by medium flow. We conclude that medium flow specifically interferes with, and up‐regulates, the activity of xenosensors and/or the expression of detoxification genes in primary human hepatocytes. Down‐regulation of detoxification genes in conventional (static) cultures is therefore partly a consequence of the absence of medium circulation.

Glucose and Fatty Acid Metabolism in a 3 Tissue In-Vitro Model Challenged with Normo- and Hyperglycaemia

Nutrient balance in the human body is maintained through systemic signaling between different cells and tissues. Breaking down this circuitry to its most basic elements and reconstructing the metabolic network in-vitro provides a systematic method to gain a better understanding of how cross-talk between the organs contributes to the whole body metabolic profile and of the specific role of each different cell type. To this end, a 3-way connected culture of hepatocytes, adipose tissue and endothelial cells representing a simplified model of energetic substrate metabolism in the visceral region was developed. The 3-way culture was shown to maintain glucose and fatty acid homeostasis in-vitro. Subsequently it was challenged with insulin and high glucose concentrations to simulate hyperglycaemia. The aim was to study the capacity of the 3-way culture to maintain or restore normal circulating glucose concentrations in response to insulin and to investigate the effects these conditions on other metabolites involved in glucose and lipid metabolism. The results show that the system’s metabolic profile changes dramatically in the presence of high concentrations of glucose, and that these changes are modulated by the presence of insulin. Furthermore, we observed an increase in E-selectin levels in hyperglycaemic conditions and increased IL-6 concentrations in insulin-free-hyperglycaemic conditions, indicating, respectively, endothelial injury and proinflammatory stress in the challenged 3-way system.

In-vitro liver model using microfabricated scaffolds in a modular bioreactor

Hepatocyte function on 3‐D microfabricated polymer scaffolds realised with the pressure‐activated microsyringe was tested under static and dynamic conditions. The dynamic cell culture was obtained using the multicompartment modular bioreactor system. Hepatocyte cell density, glucose consumption, and albumin secretion rate were measured daily over a week. Cells seeded on scaffolds showed an increase in cell density compared with monolayer controls. Moreover, in dynamic culture, cell metabolic function increased three times in comparison with static monolayer cultures. These results suggest that cell density and cell‐cell interactions are mediated by the architecture of the substrate, while the endogenous biochemical functions are regulated by a sustainable supply of nutrients and interstitial‐like flow. Thus, a combination of 3‐D scaffolds and dynamic flow conditions are both important for the development of a hepatic tissue model for applications in drug testing and regenerative medicine.

A flexible bioreactor system for constructing in vitro tissue and organ models.

To develop in vitro models of cells, tissues and organs we have designed and realized a series of cell culture chambers. Each chamber is purpose designed to simulate a particular feature of the in vivo environment. The bioreactor system is user friendly, and the chambers are easy to produce, sterilize and assemble. In addition they can be connected together to simulate inter‐organ or tissue cross‐talk. Here we discuss the design philosophy of the bioreactor system and then describe its construction. Preliminary results of validation tests obtained with hepatocytes and endothelial cells are also reported. The results show that endothelial cells are extremely sensitive to small levels of shear stress and that the presence of heterotypic signals from endothelial cells enhances the endogenous metabolic function of hepatocytes. Biotechnol. Bioeng. 2011;108:2129–2140.

Flow‐regulated glucose and lipid metabolism in adipose tissue, endothelial cell and hepatocyte cultures in a modular bioreactor

Static cell culture has serious limitations in its ability to represent cellular behaviour within a live organism. In vivo, cells are constantly exposed to the flow of bodily fluids and contact with other cell types. Bioreactors provide the opportunity to study cells in an environment that more closely resembles the in vivo setting because cell cultures can be exposed to dynamic flow in contact with or in proximity to other cell types. In this study we compared the metabolic profile of a dynamic cell culture system to that of a static cell culture in three different cellular phenotypes: adipocytes, endothelial cells and hepatocytes. Albumin, glucose, free fatty acids, glycerol, and lactate were measured over 48 h. We show that all three cell types have increased glucose uptake in the presence of flow; lactate release was also significantly affected. We provide robust evidence that the presence of flow significantly modifies cellular metabolism. While flow provides a more uniform nutrient distribution and increases metabolite turnover, our results indicate that different cell types have specific metabolic responses to flow, suggesting cell‐specific flow‐regulated activation of metabolite signalling pathways.

An in vitro model of glucose and lipid metabolism in a multicompartmental bioreactor

The energy balance in vivo is maintained through inter‐organ cross‐talk involving several different tissues. As a first step towards recapitulating the metabolic circuitry, hepatocytes, endothelial cells and adipose tissue were connected in a multicompartmental modular bioreactor to reproduce salient aspects of glucose and lipid metabolism in vitro. We first examined how the two‐way cellular interplay between adipose tissue and endothelial cells affects glucose and lipid metabolism. The hepatocyte cell line HepG2 was then added to the system, creating a three‐way connected culture, to determine whether circulating metabolite concentrations were normalized, or whether metabolic shifts, which may arise when endothelial cells and adipose tissue are placed in connection, were corrected. The addition of hepatocytes to the system prevented the drop in the concentrations of glucose, L‐alanine and lactate, and the rise in that of free fatty acids. There was no significant change in glycerol levels in either of the connected cultures. The results show that connected cultures recapitulate complex physiological systemic processes, such as glucose and lipid metabolism, and that the HepG2 hepatocytes normalize circulating metabolites in this in vitro environment in the presence of other cell types.

HEMET: Mathematical model of biochemical pathways for simulation and prediction of HEpatocyte METabolism

Many computer studies and models have been developed in order to simulate cell biochemical pathways. The difficulty of integrating all the biochemical reactions that occur in a cell in a single model is the main reason for the poor results in the prediction and simulation of cell behaviour under different chemical and physical stimuli. In this paper we have translated biochemical reactions into differential equations for the development of modular model of metabolism of a hepatocyte cultured in static and standard conditions (in a plastic multiwell placed in an incubator at 37 degrees C with 5% of CO(2)). Using biochemical equations and energetic considerations a set of non-linear differential equations has been derived and implemented in Simulink. This set of equations mimics some of the principal metabolic pathways of biomolecules present in the culture medium. The software platform developed is subdivided into separate modules, each one describing a different metabolic pathway; they constitute a library which can be used for developing new modules and models to project, predict and validate cell behaviour in vitro.

A new library of HEMET model: Insulin effects on hepatic metabolism

Prediction and simulation of cell culture behaviour, under different chemical and physical stimuli by a mathematical model, represent an innovative way to create a virtual cell laboratory, where it is possible to perform and optimize experimental protocol, saving time and money. In silico experiments permit to reproduce pathological and physiological situations and make toxicological tests. In this paper we introduce a new library of HEMET (HEpatocyte METabolism) software that allows the insulin effects on hepatic metabolism to be simulated. This new set of nonlinear differential equations, derived from biochemical reactions which involve this pancreatic hormone, allows the catabolites concentration in hepatic cell culture after insulin infusion to be predicted. The validation procedures were carried out using data obtained from specifically designed cell experiments and from literature. A user friendly interface allows to easily change model parameters, rate constants and inputs simulating a wide range of physiological and pathological scenarios.

Development of a liver model using PAM scaffolds in static and dynamic conditions

The aim of the present work was to test the functionality of hepatocytes cultured on three-dimensional polymeric scaffolds realised with pressure activated microsyringe (PAM) system in static and dynamic conditions. The dynamic cell culture conditions have been produced using a bioreactor system developed and patented by our group. Hepatocytes used in this study were HepG2 cell line. Cells were seeded and cultured on hexagonal-three-dimensional polymeric poly-lactic-co-glycolic-acid (PLGA) scaffolds, initially sterilized and functionalised with murine collagen type IV. Hepatocytes cell density, glucose consumption, albumin and transferrin secretion rate have been measured daily for the whole length (7 days) of experiment. Cells under dynamic conditions showed an increase in cell density. Moreover, the metabolic cell function increased three times in comparison with monolayer culture. These results have suggested that the combination of three-dimensional scaffolds with a well defined topology and dynamic conditions were important for the development of a hepatic tissue model that could be use for drug testing and regenerative medicine.