Expansion properties of Alginate beads as cell carrier in the fluidized bed bioartificial liver

The homogeneous expansion behaviour of liquid-fluidized beds is exploited in various fields such as minerals engineering and biotechnology. Innovative fluidized bed bioreactor concepts have been also explored for applications as bioartificial organs, particularly the bioartificial liver (1). It has been shown that the fluidized bed bioreactor constituted of alginate beads hosting liver cells is one of the promising solution to a bioartificial liver. Compared to other solutions, fluidization of alginate beads containing the cells does not suffer from the severe limitations to mass transfer between the beads and the perfusion medium. In the present work, appropriate alginate beads were prepared by the alginate drop gelation in calcium chloride. The beads were characterized in terms of size distribution and density. Sauter mean diameter of 813 m and density of 1020 kg/m3 were obtained. The latter shows a value very close to usual perfusion fluid, which required also careful evaluation of the liquid properties. Expansion properties were evaluated for free alginate beads (i.e. without hepatic cells) using saline solutions as fluidization medium. Bed expansions have been conducted in a small-size 1-cm diameter column used for perfusion in in vitro experiments as well as in a bigger 10-cm diameter column close to human size bioreactor. Velocity-voidage plots are reported and elaborated in terms of Richardson-Zaki parameters, showing the effect of walls and the different distributor. ACKNOLEDGEMENTS The financial support of the European Union through the Project FP7-PEOPLE-2012-ITN “Training network for developing innovative bioartificial devices for treatment of kidney and liver disease” is gratefully acknowledged. REFERENCES Gautier A., Carpentier B., Dufresne M., Vu Dinh Q., Paullier P., Legallais C. Impact of alginate type and bead diameter on mass transfers and the metabolic activities of encapsulated C3A cells in bioartificial liver applications. European Cells and Materials 2011, 21:94-106

Merkel Cells as Putative Regulatory Cells in Skin Disorders: An In Vitro Study

Merkel cells (MCs) are involved in mechanoreception, but several lines of evidence suggest that they may also participate in skin disorders through the release of neuropeptides and hormones. In addition, MC hyperplasias have been reported in inflammatory skin diseases. However, neither proliferation nor reactions to the epidermal environment have been demonstrated. We established a culture model enriched in swine MCs to analyze their proliferative capability and to discover MC survival factors and modulators of MC neuroendocrine properties. In culture, MCs reacted to bFGF by extending outgrowths. Conversely, neurotrophins failed to induce cell spreading, suggesting that they do not act as a growth factor for MCs. For the first time, we provide evidence of proliferation in culture through Ki-67 immunoreactivity. We also found that MCs reacted to histamine or activation of the proton gated/osmoreceptor TRPV4 by releasing vasoactive intestinal peptide (VIP). Since VIP is involved in many pathophysiological processes, its release suggests a putative regulatory role for MCs in skin disorders. Moreover, in contrast to mechanotransduction, neuropeptide exocytosis was Ca2+-independent, as inhibition of Ca2+ channels or culture in the absence of Ca2+ failed to decrease the amount of VIP released. We conclude that neuropeptide release and neurotransmitter exocytosis may be two distinct pathways that are differentially regulated

Are Co-Culture Approaches Able to Improve Biological Functions of Bioartificial Livers?

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Alginate-Based Cell Microencapsulation for Tissue Engineering and Regenerative Medicine

International audienceIncreasing numbers of requests for transplantable organs and their scarcity has led to a pressing need to find alternative solutions to standard transplantation. An appealing but challenging proposal came from the fields of tissue engineering and regenerative medicine, the purpose of which is to build tissues/organs from scratch in the laboratory and use them as either permanent substitutes for direct implantation into the patient's body, or as temporary substitutes to bridge patients until organ regeneration or transplantation. Using bioartificial constructs requires administration of immunosuppressant therapies to prevent rejection by the recipient. Microencapsulation has been identified as promising technology for immunoisolating biological materials from immune system attacks by the patient. It is based on entrapping cellular material within a spherical semipermeable polymeric scaffold. This latter defines the boundary between the internal native-like environment and the external "aggressive" one. The scaffold thus acts like a selective filter that makes possible an appropriate supply of nutrients and oxygen to the cellular constructs, while blocking the passage for adverse molecules. Alginate, which is a natural polymer, is the main biomaterial used in this context. Its excellent properties and mild gelation ability provide suitable conditions for supporting viability and preserving the functionalities of the cellular-engineered constructs over long periods. Although much remains to be done before bringing microencapsulated constructs into clinical practice, an increasing number of applications for alginate-based microencapsulation in numerous medical areas confirm the considerable potential for this technology in providing a cure for transplant in patients that excludes immunosuppressive therapies

The whole epidermis as the forefront of the sensory system

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Optimizing the fluidized bed bioreactor as an external bioartificial liver

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Rat Merkel cells are mechanoreceptors and osmoreceptors.

Merkel cells (MCs) associated with nerve terminals constitute MC-neurite complexes, which are involved in slowly-adapting type I mechanoreception. Although MCs are known to express voltage-gated Ca2+ channels and hypotonic-induced membrane deformation is known to lead to Ca2+ transients, whether MCs initiate mechanotransduction is currently unknown. To answer to this question, rat MCs were transfected with a reporter vector, which enabled their identification.Their properties were investigated through electrophysiological studies. Voltage-gated K+, Ca2+ and Ca2+-activated K+ (KCa)channels were identified, as previously described. Here, we also report the activation of Ca2+ channels by histamine and their inhibition by acetylcholine. As a major finding, we demonstrated that direct mechanical stimulations induced strong inward Ca2+ currents in MCs. Depolarizations were dependent on the strength and the length of the stimulation. Moreover, touch-evoked currents were inhibited by the stretch channel antagonist gadolinium. These data confirm the mechanotransduction capabilities of MCs. Furthermore, we found that activation of the osmoreceptor TRPV4 in FM1-43-labeled MCs provoked neurosecretory granule exocytosis. Since FM1-43 blocks mechanosensory channels, this suggests that hypo-osmolarity activates MCs in the absence of mechanotransduction. Thus, mechanotransduction and osmoreception are likely distinct pathways

Microencapsulated Hepatocytes Differentiated from Human Induced Pluripotent Stem Cells: Optimizing 3D Culture for Tissue Engineering Applications

Liver cell therapy and in vitro models require functional human hepatocytes, the sources of which are considerably limited. Human induced pluripotent stem cells (hiPSCs) represent a promising and unlimited source of differentiated human hepatocytes. However, when obtained in two-dimensional (2D) cultures these hepatocytes are not fully mature and functional. As three-dimensional culture conditions offer advantageous strategies for differentiation, we describe here a combination of three-dimensional (3D) approaches enabling the successful differentiation of functional hepatocytes from hiPSCs by the encapsulation of hiPSC-derived hepatoblasts in alginate beads of preformed aggregates. The resulting encapsulated and differentiated hepatocytes (E-iHep-Orgs) displayed a high level of albumin synthesis associated with the disappearance of α-fetoprotein (AFP) synthesis, thus demonstrating that the E-iHep-Orgs had reached a high level of maturation, similar to that of adult hepatocytes. Gene expression analysis by RT-PCR and immunofluorescence confirmed this maturation. Further functional assessments demonstrated their enzymatic activities, including lactate and ammonia detoxification, as well as biotransformation activities of Phase I and Phase II enzymes. This study provides proof of concept regarding the benefits of combining three-dimensional techniques (guided aggregation and microencapsulation) with liver differentiation protocols as a robust approach to generate mature and functional hepatocytes that offer a permanent and unlimited source of hepatocytes. Based on these encouraging results, our combined conditions to produce mature hepatocytes from hiPSCs could be extended to liver tissue engineering and bioartificial liver (BAL) applications at the human scale for which large biomasses are mandatory

Utilization of a mouse/human chimeric model for long term metabolic testing of human skin

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Effects of sangre de drago in an in vitro model of cutaneous neurogenic inflammation.

International audienceSangre de drago (SD) is a viscous bright red resin collected from Croton lechleri trees that grow in the South American jungle. This sap is used extensively in the native pharmacopoeia to treat skin disorders. Its effectiveness as an inhibitor of neurogenic inflammation has been recently demonstrated. To understand the underlying mechanisms of these effects, we examined the ability of SD to reduce substance P (SP) release in an in vitro model of cutaneous neurogenic inflammation (CNI). This model is based on an enzyme immunoassay of SP (an inducer of CNI) in a porcine co-culture of dorsal root ganglion neurons and keratinocytes. After incubation with different concentrations of SD, we noted an immediate and significant dose-dependent decrease in basal SP release, with average values of 32% at 1% SD (v/v) and 26% at 0.1% (v/v). On the other hand, pretreatment (72 or 1 h) of the co-culture with 1% SD (v/v) was sufficient to induce a 111% (72 h) or 65% (1 h) inhibition of capsaicin-induced SP release, while 0.1% SD (v/v) triggered a 109% (72 h) or 30% (1 h) inhibition. We conclude that sangre de drago is a potent inhibitor of CNI through direct inhibition of neuropeptide release by sensory afferent nerves