Bioreactor Technologies for Enhanced Organoid Culture.

An organoid is a 3D organization of cells that can recapitulate some of the structure and function of native tissue. Recent work has seen organoids gain prominence as a valuable model for studying tissue development, drug discovery, and potential clinical applications. The requirements for the successful culture of organoids in vitro differ significantly from those of traditional monolayer cell cultures. The generation and maturation of high-fidelity organoids entails developing and optimizing environmental conditions to provide the optimal cues for growth and 3D maturation, such as oxygenation, mechanical and fluidic activation, nutrition gradients, etc. To this end, we discuss the four main categories of bioreactors used for organoid culture: stirred bioreactors (SBR), microfluidic bioreactors (MFB), rotating wall vessels (RWV), and electrically stimulating (ES) bioreactors. We aim to lay out the state-of-the-art of both commercial and in-house developed bioreactor systems, their benefits to the culture of organoids derived from various cells and tissues, and the limitations of bioreactor technology, including sterilization, accessibility, and suitability and ease of use for long-term culture. Finally, we discuss future directions for improvements to existing bioreactor technology and how they may be used to enhance organoid culture for specific applications.

Nutraceuticals Synergistically Promote Osteogenesis in Cultured 7F2 Osteoblasts and Mitigate Inhibition of Differentiation and Maturation in Simulated Microgravity.

Microgravity is known to impact bone health, similar to mechanical unloading on Earth. In the absence of countermeasures, bone formation and mineral deposition are strongly inhibited in Space. There is an unmet need to identify nutritional countermeasures. Curcumin and carnosic acid are phytonutrients with anticancer, anti-inflammatory, and antioxidative effects and may exhibit osteogenic properties. Zinc is a trace element essential for bone formation. We hypothesized that these nutraceuticals could counteract the microgravity-induced inhibition of osteogenic differentiation and function. To test this hypothesis, we cultured 7F2 murine osteoblasts in simulated microgravity (SMG) in a Random Positioning Machine in the presence and absence of curcumin, carnosic acid, and zinc and evaluated cell proliferation, function, and differentiation. SMG enhanced cell proliferation in osteogenic medium. The nutraceuticals partially reversed the inhibitory effects of SMG on alkaline phosphatase (ALP) activity and did not alter the SMG-induced reduction in the expression of osteogenic marker genes in osteogenic medium, while they promoted osteoblast proliferation and ALP activity in the absence of traditional osteogenic media. We further observed a synergistic effect of the intermix of the phytonutrients on ALP activity. Intermixes of phytonutrients may serve as convenient and effective nutritional countermeasures against bone loss in space.

Anti-angiogenic activities of snake venom CRISP isolated from Echis carinatus sochureki.

BACKGROUND: Cysteine-rich secretory protein (CRISP) is present in majority of vertebrate including human. The physiological role of this protein is not characterized. We report that a CRISP isolated from Echis carinatus sochureki venom (ES-CRISP) inhibits angiogenesis. METHODS: The anti-angiogenic activity of purified ES-CRISP from snake venom was investigated in vitro using endothelial cells assays such as proliferation, migration and tube formation in Matrigel, as well as in vivo in quail embryonic CAM system. The modulatory effect of ES-CRISP on the expression of major angiogenesis factors and activation of angiogenesis pathways was tested by qRT-PCR and Western blot. RESULTS: The amino acid sequence of ES-CRISP was found highly similar to other members of this snake venom protein family, and shares over 50% identity with human CRISP-3. ES-CRISP supported adhesion to endothelial cells, although it was also internalized into the cytoplasm in a granule-like manner. It blocked EC proliferation, migration and tube formation in Matrigel. In the embryonic quail CAM system, ES-CRISP abolished neovascularization process induced by exogenous growth factors (bFGF, vpVEGF) and by developing gliomas. CRISP modulates the expression of several factors at the mRNA level, which were characterized as regulators of angiogenesis and blocked activation of MAPK Erk1/2 induced by VEGF. CONCLUSIONS: ES-CRISP was characterized as a negative regulator of the angiogenesis, by direct interaction with endothelial cells. GENERAL SIGNIFICANCE: The presented work may lead to the development of novel angiostatic therapy, as well as contribute to the identification of the physiological relevance of this functionally uncharacterized protein.

Revascularization of decellularized lung scaffolds: principles and progress.

There is a clear unmet clinical need for novel biotechnology-based therapeutic approaches to lung repair and/or replacement, such as tissue engineering of whole bioengineered lungs. Recent studies have demonstrated the feasibility of decellularizing the whole organ by removal of all its cellular components, thus leaving behind the extracellular matrix as a complex three-dimensional (3D) biomimetic scaffold. Implantation of decellularized lung scaffolds (DLS), which were recellularized with patient-specific lung (progenitor) cells, is deemed the ultimate alternative to lung transplantation. Preclinical studies demonstrated that, upon implantation in rodent models, bioengineered lungs that were recellularized with airway and vascular cells were capable of gas exchange for up to 14 days. However, the long-term applicability of this concept is thwarted in part by the failure of current approaches to reconstruct a physiologically functional, quiescent endothelium lining the entire vascular tree of reseeded lung scaffolds, as inferred from the occurrence of hemorrhage into the airway compartment and thrombosis in the vasculature in vivo. In this review, we explore the idea that successful whole lung bioengineering will critically depend on 1) preserving and/or reestablishing the integrity of the subendothelial basement membrane, especially of the ultrathin respiratory membrane separating airways and capillaries, during and following decellularization and 2) restoring vascular physiological functionality including the barrier function and quiescence of the endothelial lining following reseeding of the vascular compartment. We posit that physiological reconstitution of the pulmonary vascular tree in its entirety will significantly promote the clinical translation of the next generation of bioengineered whole lungs.

Mesenchymal stem cells for therapeutic applications in pulmonary medicine.

INTRODUCTION: Mesenchymal stem cells (MSCs) of different biological sources are in Phase 1 clinical trials and are being considered for Phase 2 therapy of lung disorders, and lung (progenitor) cells derived from pluripotent stem cells (SCs) are under development in preclinical animal models. SOURCES OF DATA: PubMed.gov and ClinicalTrials.gov. AREAS OF AGREEMENT: There is consensus about the therapeutic potential of transplanted SCs, mainly MSCs, primarily involves paracrine 'bystander' effects that confer protection of the epithelial and endothelial linings of the lung caused by inflammation and/or fibrosis and lead to increased survival in animal models. Clinical trials of Phase 1 indicate safety and suggest that the efficacy of SC therapy in patients with various lung diseases will require a higher dosage than previously evaluated. AREAS OF CONTROVERSY: A growing interest in the re-epithelialization and re-endothelialization of damaged lung tissue involves the putative pulmonary differentiation of exogenous MSCs. Currently, it is not clear whether or not the observed regeneration of distal airways/vasculature is derived from lung-resident and/or transplanted SCs. GROWING POINTS: Important topics under investigation include optimization of the cell source with a decrease in cell population heterogeneity characterized by defined markers, route of delivery for effective treatment, potential dose and therapeutic protocol of SC application, development of quantitative assays and biomarkers of lung disease and repair, and the potential use of tissue engineered lung. AREAS TIMELY FOR DEVELOPING RESEARCH: Ability of MSCs to differentiate into epithelial cells of the lung, use of autologous induced pluripotent SCs (iPSCs) derived from the patients, complete biochemical characterization of the secretome of SCs used for therapy, and the incorporation of simultaneous and/or subsequent treatment with drugs which also aid in lung repair and regeneration. CAUTIONARY NOTE: Although safety of MSC-based cell therapy was proved in Phase 1, efficacy, long-term survival and preservation of lung respiratory function need to be further evaluated, cautioning against hastily translating SCs therapy from animal models of lung injury to clinical trials of patients with lung disorders.

Tissue factor activity and ECM-related gene expression in human aortic endothelial cells grown on electrospun biohybrid scaffolds.

All blood vessels are lined with a quiescent endothelium, which aids in regulating regular blood flow and avoiding thrombus formation. Current attempts at replacing diseased blood vessels frequently fail due to the intrinsic thrombogenicity of the materials used as vascular grafts. In extending our previous work where we introduced a new candidate scaffolds for vascular grafts electrospun from a blend solution of PLGA, gelatin, and elastin (PGE), this study aimed to evaluate the potential of PGE scaffolds to support nonthrombogenic monolayers of primary isolates of human aortic endothelial cells (HAECs), as assessed by a combination of biochemical, molecular, and bioinformatics-based analyses. After 24 h of culture on 3-D fibrous PGE scaffolds, HAECs formed a confluent, nonthrombogenic, and physiologically competent monolayer, as assessed by tissue factor (TF) gene expression and protein activity assays. The levels of TF mRNA/protein activity in HAECs grown on PGE scaffolds were similar to those on gelatin or collagen IV-coated 2-D surfaces. In addition, bioinformatics-based analysis of a focused microarray containing 84 ECM-related cDNA probes demonstrated that HAECs essentially expressed a histotypic ECM-related "transcriptome" on PGE scaffolds, where cells were more quiescent than cells cultured on 2-D coverslips coated with gelatin (a well-known "inert" substrate for conventional EC culture), but less so than on 2-D PGE films. These data suggest an important role for nanorough substrates (PGE films) in passivating endothelial cells and confirm the crucial effect of substrate composition in this process. Principal component analysis of microarray data on the above substrates (including collagen IV) implied that substrate composition plays a greater role than surface topography in affecting the endothelial ECM-related "transcriptome". Taken together, our findings suggest that electrospun PGE scaffolds are potentially suitable for application in small diameter vascular tissue engineering.

Electrospun rapamycin-eluting polyurethane fibers for vascular grafts.

PURPOSE: To develop rapamycin-eluting electrospun polyurethane (PU) vascular grafts that could effectively suppress local smooth muscle cell (SMC) proliferation. METHODS: Rapamycin (RM) was incorporated in PU fibers by blend electrospinning using three distinct blending methods. The drug release profiles and the bioavailability of RM-containing PU fibers in the form of fibrous mats and vascular grafts were evaluated up to 77 days in vitro. RESULTS: RM-contained PU fibers generated by the three distinct blending methods exhibited significantly different fiber diameters (200-500 nm) and distinct RM release kinetics. Young's moduli of the electrospun fibrous mats increased with higher RM contents and decreased with larger fiber diameters. For all blending methods, RM release kinetics was characteristic of a Fickian diffusion for at least 77 days in vitro. RM-PU fibers generated via powder blending showed the highest encapsulation efficiency. The RM in grafts made of these fibers remained bioactive and was still able to inhibit smooth muscle cell proliferation after 77 days of continual in vitro release. CONCLUSIONS: Electrospun RM-containing PU fibers can serve as effective drug carriers for the local suppression of SMC proliferation and could be used as RM-eluting scaffolds for vascular grafts.

NGF promotes hemodynamic recovery in a rabbit hindlimb ischemic model through trkA- and VEGFR2-dependent pathways.

Nerve growth factor (NGF) has been reported to play an important role in physiological and pathological angiogenesis. Based on these observations, we hypothesized that NGF may induce the formation of functional blood vessels in a hindlimb ischemic rabbit model. Hindlimb ischemia was induced in 34 rabbits bilaterally by endovascular embolization of femoral arteries. On the 7th, 14th, and 20th postembolization days, NGF was injected intramuscularly, in 1 ischemic limb, and vehicle was injected in the contralateral control limb. On the 40th day, newly developed collateral vessels (diameter >500 µm) were quantified by transauricular intraarterial subtraction angiography. Perfusion analysis of an in vivo dynamic computed tomography study was performed to the limbs to investigate the hemodynamic recovery of the distal ischemic tissues. Functional estimation of limb perfusion showed a statistically significant increase of blood flow and blood volume for NGF. However, the increase of the collateral vessels was not detectable angiographically, providing evidence for the existence of a NGF-stimulated capillary angiogenic network but not increase of arteriogenesis. The combination of NGF with either tropomyosin-related kinase type A or vascular endothelial growth factor receptor 2 antagonists abolished the NGF-induced hemodynamic recovery. These findings provide new insights into understanding the involvement of NGF in vascular formation and its applications in therapeutic angiogenesis.

Neural stem cells: therapeutic potential for neurodegenerative diseases.

INTRODUCTION: Neural stem cells (NSCs) from specific brain areas or developed from progenitors of different sources are of therapeutic potential for neurodegenerative diseases. SOURCES OF DATA: Treatment strategies involve the (i) transplantation of exogenous NSCs; (ii) pharmacological modulations of endogenous NSCs and (iii) modulation of endogenous NSCs via the transplantation of exogenous NSCs. AREAS OF AGREEMENT: There is a consensus about the therapeutic potential of transplanted NSCs. The ability of NSCs to home into areas of central nervous system injury allows their delivery by intravenous injection. There is also a general agreement about the neuroprotective mechanisms of NSCs involving a 'bystander effect'. AREAS OF CONTROVERSY: Individual laboratories may be using phenotypically diverse NSCs, since these cells have been differentiated by a variety of neurotrophins and/or cultured on different ECM proteins, therefore differing in the expression of neuronal markers. GROWING POINTS: Optimization of the dose, delivery route, timing of administration of NSCs, their interactions with the immune system and combination therapies in conjunction with tissue engineered neural prostheses are under investigation. AREAS TIMELY FOR DEVELOPING RESEARCH: In-depth understanding of the biological properties of NSCs, including mechanisms of therapy, safety, efficacy and elimination from the organism. These areas are central for further use in cell therapy. CAUTIONARY NOTE: As long as critical safety and efficacy issues are not resolved, we need to be careful in translating NSC therapy from animal models to patients.

Impairment of 7F2 osteoblast function by simulated partial gravity in a Random Positioning Machine.

The multifaceted adverse effects of reduced gravity pose a significant challenge to human spaceflight. Previous studies have shown that bone formation by osteoblasts decreases under microgravity conditions, both real and simulated. However, the effects of partial gravity on osteoblasts' function are less well understood. Utilizing the software-driven newer version of the Random Positioning Machine (RPMSW), we simulated levels of partial gravity relevant to future manned space missions: Mars (0.38 G), Moon (0.16 G), and microgravity (Micro, ~10-3 G). Short-term (6 days) culture yielded a dose-dependent reduction in proliferation and the enzymatic activity of alkaline phosphatase (ALP), while long-term studies (21 days) showed a distinct dose-dependent inhibition of mineralization. By contrast, expression levels of key osteogenic genes (Alkaline phosphatase, Runt-related Transcription Factor 2, Sparc/osteonectin) exhibited a threshold behavior: gene expression was significantly inhibited when the cells were exposed to Mars-simulating partial gravity, and this was not reduced further when the cells were cultured under simulated Moon or microgravity conditions. Our data suggest that impairment of cell function with decreasing simulated gravity levels is graded and that the threshold profile observed for reduced gene expression is distinct from the dose dependence observed for cell proliferation, ALP activity, and mineral deposition. Our study is of relevance, given the dearth of research into the effects of Lunar and Martian gravity for forthcoming space exploration.

Co-electrospun blends of PLGA, gelatin, and elastin as potential nonthrombogenic scaffolds for vascular tissue engineering.

In search for novel biomimetic scaffolds for application in vascular tissue engineering, we evaluated a series of fibrous scaffolds prepared by coelectrospinning tertiary blends of poly(lactide-co-glycolide) (PLGA), gelatin, and elastin (PGE). By systematically varying the ratios of PLGA and gelatin, we could fine-tune fiber size and swelling upon hydration as well as the mechanical properties of the scaffolds. Of all PGE blends tested, PGE321 (PLGA, gelatin, elastin v/v/v ratios of 3:2:1) produced the smallest fiber size (317 ± 46 nm, 446 ± 69 nm once hydrated) and exhibited the highest Young's modulus (770 ± 131 kPa) and tensile strength (130 ± 7 kPa). All PGE scaffolds supported the attachment and metabolization of human endothelial cells (ECs) and bovine aortic smooth muscle cells (SMCs) with some variances in EC morphology and cytoskeletal spreading observed at 48 h postseeding, whereas no morphologic differences were observed at confluence (day 8). The rate of metabolization of ECs, but not of SMCs, was lower than that on tissue culture plastic and depended on the specific PGE composition. Importantly, PGE scaffolds were capable of guiding the organotypic distribution of ECs and SMCs on and within the scaffolds, respectively. Moreover, the EC monolayer generated on the PGE scaffold surface was nonthrombogenic and functional, as assessed by the basal and cytokine-inducible levels of mRNA expression and amidolytic activity of tissue factor, a key player in the extrinsic clotting cascade. Taken together, our data indicate the potential application of PGE scaffolds in vascular tissue engineering.

Cytotoxicity tests of water soluble ZnS and CdS quantum dots.

Cytotoxicity tests of zinc sulfide (ZnS) and cadmium sulfide (CdS) quantum dots (QDs) synthesized via all-aqueous process with various surface conditions were carried out with human endothelial cells (EA hy926) using two independent viability assays, i.e., by cell counting following Trypan blue staining and by measuring Alamar Blue (AB) fluorescence. The ZnS QDs with all four distinct types of surface conditions were nontoxic at both 1 microM and 10 microM concentrations for at least 6 days. On the other hand, the CdS QDs were nontoxic only at 1 microM, and showed significant cytotoxicity at 10 microM after 3 days in the cell counting assay and after 4 days in the AB fluorescence assay. The CdS QDs with (3-mercaptopropyl)trimethoxysilane (MPS)-replacement plus silica capping were less cytotoxic than those with 3-mercaptopropionic acid (MPA) capping and those with MPS-replacement capping. Comparing the results of ZnS and CdS QDs with the same particle size, surface condition and concentration, it is indicated that the cytotoxicity of CdS QDs and the lack of it in ZnS QDs were probably due to the presence and absence of the toxic Cd element, respectively. The nontoxicity of the aqueous ZnS QDs makes them favorable for in vivo imaging applications.

Neurotrophic factors and their receptors in lung development and implications in lung diseases.

Although lung innervation has been described by many studies in humans and rodents, the regulation of the respiratory system induced by neurotrophins is not fully understood. Here, we review current knowledge on the role of neurotrophins and the expression and function of their receptors in neurogenesis, vasculogenesis and during the embryonic development of the respiratory tree and highlight key implications relevant to respiratory diseases.

On the road to smart biomaterials for bone research: definitions, concepts, advances, and outlook.

The demand for biomaterials that promote the repair, replacement, or restoration of hard and soft tissues continues to grow as the population ages. Traditionally, smart biomaterials have been thought as those that respond to stimuli. However, the continuous evolution of the field warrants a fresh look at the concept of smartness of biomaterials. This review presents a redefinition of the term "Smart Biomaterial" and discusses recent advances in and applications of smart biomaterials for hard tissue restoration and regeneration. To clarify the use of the term "smart biomaterials", we propose four degrees of smartness according to the level of interaction of the biomaterials with the bio-environment and the biological/cellular responses they elicit, defining these materials as inert, active, responsive, and autonomous. Then, we present an up-to-date survey of applications of smart biomaterials for hard tissues, based on the materials' responses (external and internal stimuli) and their use as immune-modulatory biomaterials. Finally, we discuss the limitations and obstacles to the translation from basic research (bench) to clinical utilization that is required for the development of clinically relevant applications of these technologies.

Multifunctional Dental Composite with Piezoelectric Nanofillers for Combined Antibacterial and Mineralization Effects.

After nearly seven decades of development, dental composite restorations continue to show limited clinical service. The triggering point for restoration failure is the degradation of the bond at the tooth-biomaterial interface from chemical, biological, and mechanical sources. Oral biofilms form at the bonded interfaces, producing enzymes and acids that demineralize hard tissues and damage the composite. Removing bacteria from bonded interfaces and remineralizing marginal gaps will increase restorations' clinical service. To address this need, we propose for the first time the use of piezoelectric nanoparticles of barium titanate (BaTiO3) as a multifunctional bioactive filler in dental resin composites, offering combined antibacterial and (re)mineralization effects. In this work, we developed and characterized the properties of dental piezoelectric resin composites, including the degree of conversion and mechanical and physical properties, for restorative applications. Moreover, we evaluated the antibacterial and mineralization responses of piezoelectric composites in vitro. We observed a significant reduction in biofilm growth (up to 90%) and the formation of thick and dense layers of calcium phosphate minerals in piezoelectric composites compared to control groups. The antibacterial mechanism was also revealed. Additionally, we developed a unique approach evaluating the bond strength of dentin-adhesive-composite interfaces subjected to simultaneous attacks from bacteria and cyclic mechanical loading operating in synergy. Our innovative bioactive multifunctional composite provides an ideal technology for restorative applications using a single filler with combined long-lasting nonrechargeable antibacterial/remineralization effects.

Pre-coating decellularized liver with HepG2-conditioned medium improves hepatic recellularization.

Liver transplantation from compatible donors has been the main therapy available for patients with irreversible hepatic injuries. Due to the increasing shortage of organs suitable for transplantation, tissue engineering technologies are important alternatives or surrogate approaches for the future of human organ transplantations. New bioengineering tools have been designed to produce decellularized organs (i.e. scaffolds) which could be recellularized with human cells. Specifically, there is an unmet need for developing reproducible protocols for inducing better cellular spreading in decellularized liver scaffolds. The aim of the present work was to investigate the possibility to improve liver scaffold recellularization by pre-coating decellularized tissue scaffolds with HepG2-conditioned medium (CM). Furthermore, we evaluated the capability of commercial human liver cells (HepG2) to adhere to several types of extracellular matrices (ECM) as well as CM components. Wistar rat livers were decellularized and analyzed by histology, scanning electron microscopy (SEM), immunohistochemistry and residual DNA-content analysis. Human induced pluripotent stem cells (hiPSCs)-derived mesenchymal cells (hiMSCs), and human commercial hepatic (HepG2) and endothelial (HAEC) cells were used for liver scaffold recellularization with or without CM pre-coating. Recellularization occurred for up to 5 weeks. Hepatic tissues and CM were analyzed by proteomic assays. We show that integrity and anatomical organization of the hepatic ECM were maintained after decellularization, and proteomic analysis suggested that pre-coating with CM enriched the decellularized liver ECM. Pre-coating with HepG2-CM highly improved liver recellularization and revealed the positive effects of liver ECM and CM components association.

Angiostatic effects of K252a, a Trk inhibitor, in murine brain capillary endothelial cells.

Nerve growth factor (NGF) supports the survival and differentiation of sympathetic and sensory neurons and is also mitogenic for a variety of tumors. K252a, an antagonist of NGF receptor TrkA, was previously used as a pharmacological tool to study NGF actions and as a lead compound for developing anti-tumor drugs. Since recently, NGF was characterized as an angiogenic factor, we sought to investigate the angiostatic properties of K252a on endothelial cells (ECs). For this purpose, we used a murine brain microcapillary ECs model in which we found autocrine release of NGF in the culture medium and activation of TrkA receptor-induced downstream signaling molecules Erk1/2, Akt, and PLCgamma. In this model, we demonstrated the angiostatic property of K252a based on its ability to affect several important angiogenic steps. K252a, but not its cell membrane impermeable analogue K252b at 100 nM: (i) inhibited the proliferation of the ECs by 45 +/- 9%; (ii) reduced by 70 +/- 4% the migration of the ECs measured in a wound-closure model; (iii) reduced by 29 +/- 9% the formation of tube-like structures of the ECs cultured on Matrigel; (iv) stimulated by 100 +/- 25% the collagen deposition by the ECs, a process responsible for the increased endothelial barrier functions expressed by 22 +/- 5% reduction of paracellular permeability and by 17 +/- 3% elevation of transendothelial electrical resistance. These data suggest that NGF/TrkA may represent a target for the development of novel, K252a-derived multikinase inhibitors drugs with anti-tumor and angiostatic dual activities.

Cell-Based Adhesion Assays for Isolation of Snake Venom's Integrin Antagonists.

Snake venoms could lead to the development of new drugs to treat a range of life-threatening conditions like cardiovascular diseases. Most snake venoms contain a large variety of lethal toxins as well as anti-adhesive proteins such as disintegrins, which have evolved from the harmless compounds ADAMs (proteins with a disintegrin and a metalloprotease domain) and C-type lectin proteins which disturb connective tissue and cell-matrix interaction. These anti-adhesive proteins target and block integrin receptors and disrupt normal biological processes in snakes' prey such as connective tissue physiology and blood clotting. This chapter provides the experimental details of a practical, cell-based adhesion protocol to help identify and isolate disintegrins and C-type lectin proteins from snake venoms, important tools in integrin research and lead compounds for drug discovery.

Effects of Fluorescent Diamond Particles FDP-NV-800nm on Essential Biochemical Functions of Primary Human Umbilical Vein Cells and Human Hepatic Cell Line, HepG-2 in vitro (Part VI): Acute Biocompatibility Studies.

BACKGROUND: Recently, we reported the safety and biocompatibility of fluorescent diamond particles, FDP-NV-Z-800nm (FDP-NV) injected intravenously into rats, where no morbidity and mortality were noted over a period of 3 months. The acute effects of FDP-NV-800nm particles on cultured human endothelial and hepatic cells remain unexplored. PURPOSE: In this study, we aimed to explore select cellular and biochemical functions in cultured human umbilical endothelial cells (HUVEC) and a human hepatic cancer cell line (HepG-2) exposed to FDP-NV-800 in vitro at exposure levels within the pharmacokinetics (Cmax and the nadir) previously reported in vivo. METHODS: Diverse cellular and biochemical functions were monitored, which cumulatively can provide insights into some vital cellular functions. Cell proliferation and migration were assessed by quantitative microscopy. Mitochondrial metabolic functions were tested by the MTT assay, and cytosolic esterase activity was studied by the calcein AM assay. Chaperons (CHOP), BiP and apoptosis (caspase-3 activation) were monitored by using Western blot (WB). MAPK Erk1/2 signaling was assessed by the detection of the phosphorylated form of the protein (P-Erk 1/2) and its translocation into the cell nucleus. RESULTS: At all concentrations tested (0.001-0.1mg/mL), FDP-NV did not affect any of the biomarkers of cell integrity of HepG2 cells. In contrast, the proliferation of HUVEC was affected at the highest concentration tested (0.1mg/mL, Cmax). Exposure of HUVEC to (0.01 mg/mL) FDP-NV had a mild-moderate effect on cell proliferation as evident in the MTT assay and was absent when proliferation was assessed by direct cell counting or by using the calcein AM assays. In both cell types, exposure to the highest concentration (0.1 mg/mL) of FDP-NV did neither affect FBS-stimulated cell signaling (MAPK Erk1/2 phosphorylation) nor did it activate of Caspase 3. CONCLUSION: Our data suggest that FDP-NV-800nm are largely biocompatible with HepG-2 cells proliferation within the pharmacokinetic data reported previously. In contrast, HUVEC proliferation at the highest exposure dose (0.1 mg/mL) responded adversely with respect to several biomarkers of cell integrity. However, since the Cmax levels are very short-living, the risk for endothelial injury is likely minimal for slow rate cell proliferation such as endothelial cells.

Enhanced Induction of Definitive Endoderm Differentiation of Mouse Embryonic Stem Cells in Simulated Microgravity.

Directed in vitro differentiation of pluripotent stem cells toward definitive endoderm (DE) offers great research and therapeutic potential since these cells can further differentiate into cells of the respiratory and gastrointestinal tracts, as well as associated organs such as pancreas, liver, and thyroid. We hypothesized that culturing mouse embryonic stem cells (mESCs) under simulated microgravity (SMG) conditions in rotary bioreactors (BRs) will enhance the induction of directed DE differentiation. To test our hypothesis, we cultured the cells for 6 days in two-dimensional monolayer colony cultures or as embryoid bodies (EBs) in either static conditions or, dynamically, in the rotary BRs. We used flow cytometry and quantitative polymerase chain reaction to analyze the expression of marker proteins and genes, respectively, for pluripotency (Oct3/4) and mesendodermal (Brachyury T), endodermal (FoxA2, Sox17, CxCr4), and mesodermal (Vimentin, Meox1) lineages. Culture in the form of EBs in maintenance media in the presence of leukemia inhibitory factor, in static or SMG conditions, induced expression of some of the differentiation markers, suggesting heterogeneity of the cells. This is in line with previous studies showing that differentiation is initiated as cells are aggregated into EBs even without supplementing differentiation factors to the media. Culturing EBs in static conditions in differentiation media (DM) in the presence of activin A reduced Oct3/4 expression and significantly increased Brachyury T and CxCr4 expression, but downregulated FoxA2 and Sox17. However, culturing in SMG BRs in DM upregulated Brachyury T and all of the DE markers and reduced Oct3/4 expression, indicating the advantage of dynamic cultures in BRs to specifically enhance directed DE differentiation. Given the potential discrepancies between the SMG conditions on earth and actual microgravity conditions, as observed in other studies, future experiments in space flight are required to validate the effects of reduced gravity on mESC differentiation.