A tan in a test tube -in vitro models for investigating ultraviolet radiation-induced damage in skin

Presently, global rates of skin cancers induced by ultraviolet radiation (UVR) exposure are on the rise. In view of this, current knowledge gaps in the biology of photocarcinogenesis and skin cancer progression urgently need to be addressed. One factor that has limited skin cancer research has been the need for a reproducible and physiologically-relevant model able to represent the complexity of human skin. This review outlines the main currently-used in vitro models of UVR-induced skin damage. This includes the use of conventional two-dimensional cell culture techniques and the major animal models that have been employed in photobiology and photocarcinogenesis research. Additionally, the progression towards the use of cultured skin explants and tissue-engineered skin constructs, and their utility as models of native skin's responses to UVR are described. The inherent advantages and disadvantages of these in vitro systems are also discussed.

In vitro models for investigating keratinocyte responses to ultraviolet B radiation

This thesis describes the use of 2- and 3-dimensional cell-based models for studying how skin cells respond to ultraviolet radiation. These methods were used to investigate skin damage and repair after exposure to radiation in the context of skin cancer development. Interactions between different skin cell types were demonstrated as being significant in protecting against ultraviolet radiation-induced skin damage. This has important implications in understanding how skin cancers occur, as well as in the development of new strategies to prevent and treat them.

In vivo and in vitro models demonstrate a role for caveolin-1 in the pathogenesis of ischaemic acute renal failure

Caveolae and their proteins, the caveolins, transport macromolecules; compartmentalize signalling molecules; and are involved in various repair processes. There is little information regarding their role in the pathogenesis of significant renal syndromes such as acute renal failure (ARF). In this study, an in vivo rat model of 30 min bilateral renal ischaemia followed by reperfusion times from 4 h to 1 week was used to map the temporal and spatial association between caveolin-1 and tubular epithelial damage (desquamation, apoptosis, necrosis). An in vitro model of ischaemic ARF was also studied, where cultured renal tubular epithelial cells or arterial endothelial cells were subjected to injury initiators modelled on ischaemia-reperfusion (hypoxia, serum deprivation, free radical damage or hypoxia-hyperoxia). Expression of caveolin proteins was investigated using immunohistochemistry, immunoelectron microscopy, and immunoblots of whole cell, membrane or cytosol protein extracts. In vivo, healthy kidney had abundant caveolin-1 in vascular endothelial cells and also some expression in membrane surfaces of distal tubular epithelium. In the kidneys of ARF animals, punctate cytoplasmic localization of caveolin-1 was identified, with high intensity expression in injured proximal tubules that were losing basement membrane adhesion or were apoptotic, 24 h to 4 days after ischaemia-reperfusion. Western immunoblots indicated a marked increase in caveolin-1 expression in the cortex where some proximal tubular injury was located. In vitro, the main treatment-induced change in both cell types was translocation of caveolin-1 from the original plasma membrane site into membrane-associated sites in the cytoplasm. Overall, expression levels did not alter for whole cell extracts and the protein remained membrane-bound, as indicated by cell fractionation analyses. Caveolin-1 was also found to localize intensely within apoptotic cells. The results are indicative of a role for caveolin-1 in ARF-induced renal injury. Whether it functions for cell repair or death remains to be elucidated.

Three dimensional in-vitro models for studying cancer angiogenesis

Introduction Hydrogels prepared from star-shaped poly(ethylene glycol) (PEG) and maleimide-functionalized heparin provide a potential matrix for use in developing three dimensional (3D) models. We have previously demonstrated that these hydrogels support the cultivation of human umbilical vein endothelial cells (HUVECs). We extend this body of work to study the ability to create an extracellular matrix (ECM)-like model to study breast and prostate cancer cell growth in 3D. Also, we investigate the ability to produce a tri-culture mimicking tumour angiogenesis with cancer spheroids, HUVECs and mesenchymal stem cells (MSCs). Materials and Methods The breast cancer cell lines, MCF-7 and MDA-MB-231, and prostate cancer cell lines, LNCaP and PC3, were seeded into starPEG-heparin hydrogels and grown for 14 Days to analyze the effects of varying hydrogel stiffness on spheroid development. Resulting hydrogel constructs were analyzed via proliferation assays, light microscopy, and immunostaining. Cancer cell lines were then seeded into starPEG-heparin hydrogels functionalized with growth factors as spheroids with HUVECs and MSCs and grown as a tri-culture. Cultures were analyzed via immunostaining and observed using confocal microscopy. Results Cultures prepared in MMP-cleavable starPEG-heparin hydrogels display spheroid formation in contrast to adherent growth on tissue culture plastic. Small differences were visualized in cancer spheroid growth between different gel stiffness across the range of cell lines. Cancer cell lines were able to be co-cultivated with HUVECs and MSC. Interaction was visualized between tumours and HUVECs via confocal microscopy. Further studies intend to further optimize and mimic the ECM environment of in-situ tumour angiogenesis. Discussion Our results confirm the suitability of hydrogels constructed from starPEG-heparin for HUVEC and MSC co-cultivation with cancer cell lines to study cell-cell and cell-matrix interactions in a 3D environment. This represents a step forward in the development of 3D culture models to study the pathomechanisms of breast and prostate cancer.

Putative neuroprotective compounds in in vitro models of dopaminergic neurodegeneration

Parkinson´s disease (PD) is a debilitating age-related neurological disorder that affects various motor skills and can lead to a loss of cognitive functions. The motor symptoms are the result of the progressive degeneration of dopaminergic neurons within the substantia nigra. The factors that influence the pathogenesis and the progression of the neurodegeneration remain mostly unclear. This study investigated the role of various programmed cell death (PCD) pathways, oxidative stress, and glial cells both in dopaminergic neurodegeneration and in the protective action of various drugs. To this end, we exposed dopaminergic neuroblastoma cells (SH-SY5Y cells) to 6-OHDA, which produces oxidative stress and activates various PCD modalities that result in neuronal degeneration. Additionally, to explore the role of glia, we prepared rat midbrain primary mixed-cell cultures containing both neurons and glial cell types such as microglia and astroglia and then exposed the cultures to either MPP plus or lipopolysaccharide. Our results revealed that 6-OHDA activated several PCD pathways including apoptosis, autophagic stress, lysosomal membrane permeabilization, and perhaps paraptosis in SH-SY5Y cells. Furthermore, we found that minocycline protected SH-SY5Y cells from 6-OHDA by inhibiting both apoptotic and non-apoptotic PCD modalities. We also observed an inconsistent neuroprotective effect of various dietary anti-oxidant compounds against 6-OHDA toxicity in vitro in SH-SY5Y cells. Specifically, quercetin and curcumin exerted neuroprotection only within a narrow concentration range and a limited time frame, whereas resveratrol and epigallocatechin 3-gallate provided no protection whatsoever. Lastly, we found that molecules such as amantadine may delay or even halt the neurodegeneration in primary cell cultures by inhibiting the release of neurotoxic factors from overactivated microglia and by enhancing the pro-survival actions of astroglia. Together these data suggest that the strategy of dampening oxidative species with anti-oxidants is less effective than preventing the production of toxic factors such as oxidative and pro-inflammatory molecules by pathologically activated microglia. This would subsequently prevent the activation of various PCD modalities that cause neuronal degeneration.

Cellular and in-vitro models to assess antioxidant activities of seaweed extracts and the potential use of the extracts as ingredients

Seaweeds contain a range of antioxidant compounds such as polyphenols, carotenoids, sulphated polysaccharides and vitamins and have the potential to be used as ingredients in neutraceuticals. The antioxidant activity of crude 60% methanol extracts prepared from five Irish seaweeds, Ascophyllum nodosum, Laminaria hyperborea, Pelvetia canaliculata, Fucus vesiculosus and Fucus serratus were examined using in-vitro assays and a cell model system to determine the antioxidant activity of the extracts and their ability to protect against H2O2 and tert-BOOH-induced DNA damage and alterations in cellular antioxidant status in the human adenocarcinoma, Caco-2 cell line. To optimise the extraction of antioxidant compounds from seaweeds, an accelerated solvent extraction (ASE®) was used in combination with food grade solvents. The antioxidant activity of these extracts against H2O2 and tert-BOOH-induced DNA damage and alterations in cellular antioxidant status was also assessed. Extracts that exhibited the highest antioxidant activity, A. nodosum (100% water and 80% ethanol extracts) and F. vesiculosus (60% ethanol extract) were selected as ingredients for incorporation into fluid milk and yogurt at concentrations of 0.25% and 0.5%. The addition of the seaweed extracts to milk and yogurt did not affect the pH or shelf-life properties of the products. Seaweed addition did however significantly influence the colour properties of the milk and yogurt. Yellowness values were significantly higher in yogurts containing F. vesiculosus at both concentrations and A. nodosum (80% ethanol) at the 0.5% concentration. In milk, the F. vesiculosus (60% ethanol) and A. nodosum (80% ethanol) at both the 0.25% and the 0.5% concentrations had higher greenness and yellowness values than the milk containing A. nodosum (100% water). Sensory analysis revealed that appearance and flavour governed the overall acceptability of yogurts with the control yogurt, and yogurts containing A. nodosum (100% water) were the most preferred samples by panellists. However, in the milk trial the perception of a fishy taste was the determining factor in the negative perception of milk. The unsupplemented control and the milk containing A. nodosum (100% water) at a concentration of 0.5% were the most overall accepted milk samples by the sensory panellists. The antioxidant activity of the extracts in milk and yogurt remained stable during storage as determined by the in-vitro assays. Seaweed supplemented milk and yogurt were also subjected to an in-vitro digestion procedure which mimics the human digestive system. The milk and yogurt samples and their digestates were added to Caco-2 cells to investigate their antioxidant potential however neither the undigested or digested samples protected against H2O2-induced DNA damage in Caco-2 cells.

Development of human central nervous system 3D in vitro models for preclinical research

Neurological disorders are a major concern in modern societies, with increasing prevalence mainly related with the higher life expectancy. Most of the current available therapeutic options can only control and ameliorate the patients’ symptoms, often be-coming refractory over time. Therapeutic breakthroughs and advances have been hampered by the lack of accurate central nervous system (CNS) models. The develop-ment of these models allows the study of the disease onset/progression mechanisms and the preclinical evaluation of novel therapeutics. This has traditionally relied on genetically engineered animal models that often diverge considerably from the human phenotype (developmentally, anatomically and physiologically) and 2D in vitro cell models, which fail to recapitulate the characteristics of the target tissue (cell-cell and cell-matrix interactions, cell polarity). The in vitro recapitulation of CNS phenotypic and functional features requires the implementation of advanced culture strategies that enable to mimic the in vivo struc-tural and molecular complexity. Models based on differentiation of human neural stem cells (hNSC) in 3D cultures have great potential as complementary tools in preclinical research, bridging the gap between human clinical studies and animal models. This thesis aimed at the development of novel human 3D in vitro CNS models by integrat-ing agitation-based culture systems and a wide array of characterization tools. Neural differentiation of hNSC as 3D neurospheres was explored in Chapter 2. Here, it was demonstrated that human midbrain-derived neural progenitor cells from fetal origin (hmNPC) can generate complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Chapter 3 focused on the development of cellular characterization assays for cell aggregates based on light-sheet fluorescence imaging systems, which resulted in increased spatial resolu-tion both for fixed samples or live imaging. The applicability of the developed human 3D cell model for preclinical research was explored in Chapter 4, evaluating the poten-tial of a viral vector candidate for gene therapy. The efficacy and safety of helper-dependent CAV-2 (hd-CAV-2) for gene delivery in human neurons was evaluated, demonstrating increased neuronal tropism, efficient transgene expression and minimal toxicity. The potential of human 3D in vitro CNS models to mimic brain functions was further addressed in Chapter 5. Exploring the use of 13C-labeled substrates and Nucle-ar Magnetic Resonance (NMR) spectroscopy tools, neural metabolic signatures were evaluated showing lineage-specific metabolic specialization and establishment of neu-ron-astrocytic shuttles upon differentiation. Chapter 6 focused on transferring the knowledge and strategies described in the previous chapters for the implementation of a scalable and robust process for the 3D differentiation of hNSC derived from human induced pluripotent stem cells (hiPSC). Here, software-controlled perfusion stirred-tank bioreactors were used as technological system to sustain cell aggregation and dif-ferentiation. The work developed in this thesis provides practical and versatile new in vitro ap-proaches to model the human brain. Furthermore, the culture strategies described herein can be further extended to other sources of neural phenotypes, including pa-tient-derived hiPSC. The combination of this 3D culture strategy with the implemented characterization methods represents a powerful complementary tool applicable in the drug discovery, toxicology and disease modeling.

Fermentation of resistant starches: influence of in vitro models on colon carcinogenesis

Resistant starch type 2 (RS2) and type 3 (RS3) containing preparations were digested using a batch (a) and a dynamic in vitro model (b). Furthermore, in vivo obtained indigestible fractions from ileostomy patients were used (c). Subsequently these samples were fermented with human feces with a batch and a dynamic in vitro method. The fermentation supernatants were used to treat CAC02 cells. Cytotoxicity, anti-genotoxicity against hydrogen peroxide (comet assay) and the effect on barrier function measured by trans-epithelial electrical resistance were determine. Dynamically fermented samples led to high cytotoxic activity, probably due to additional compounds added during in vitro fermentation. As a consequence only batch fermented samples were investigated further. Batch fermentation of RS resulted in an anti-genotoxic activity ranging from 9-30% decrease in DNA damage for all the samples, except for RS2-b. It is assumed that the changes in RS2 structures due to dynamic digestion resulted in a different fermentation profile not leading to any anti-genotoxic effect. Additionally, in vitro batch fermentation of RS caused an improvement in integrity across the intestinal barrier by approximately 22% for all the samples. We have demonstrated that batch in vitro fermentation of RS2 and RS3 preparations differently pre-digested are capable of inhibiting the initiation and promotion stage in colon carcinogenesis in vitro.

A comparison of the anticancer properties of isoxanthohumol and 8-prenylnaringenin using in vitro models of colon cancer

The hops plant (Humulus lupulus L.) is an essential ingredient in beer and contains a number of potentially bioactive prenylflavonoids, the predominant being the weakly estrogenic isoxanthohumol (Ix), which can be converted to the more strongly estrogenic 8-PN by the colonic microbiota. The aim of this study was to investigate the biological activity of 8-PN and Ix using in vitro models representing key stages of colorectal carcinogenesis, namely cell growth and viability (MTT assay), cell-cycle progression (DNA content assay), DNA damage (Comet assay), and invasion (Matrigel assay). A significant decrease in Caco-2 cell viability was noted after both 8-PN and Ix treatments at the higher doses (40 and 50 μM, respectively) although the impact on cell cycle differed between the two compounds. The decreased cell viability observed after Ix treatment was associated with a concentration-dependent increase in G2/M and an increased sub-G1 cell-cycle fraction, whereas treatment with 8-PN was associated with an elevated G0/G1 and an increased sub-G1 cell-cycle fraction. Significant antigenotoxic activity was noted at all 8-PN concentrations tested (5-40 μM). Although significant antigenotoxic activity was also noted with Ix treatment at ≤25 μM, at a higher dose, Ix itself exerted genotoxic activity. In a dose-dependent manner, both compounds inhibited HT115 cell invasion with reductions up to 52 and 46% for Ix and 8-PN, respectively, in comparison to untreated cells. This study demonstrated that both Ix and its gut microbial metabolite 8-PN exert anticancer effects on models of key stages of colon tumourigenesis.

Protective effects of the galectin-1 protein on in vivo and in vitro models of ocular inflammation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

In vitro models of the human epithelial airway barrier to study the toxic potential of particulate matter

BACKGROUND: Several epidemiological studies show that inhalation of particulate matter may cause increased pulmonary morbidity and mortality. Of particular interest are the ultrafine particles that are particularly toxic. In addition more and more nanoparticles are released into the environment; however, the potential health effects of these nanoparticles are yet unknown. OBJECTIVES: To avoid particle toxicity studies with animals many cell culture models have been developed during the past years. METHODS: This review focuses on the most commonly used in vitro epithelial airway and alveolar models to study particle-cell interactions and particle toxicity and highlights advantages and disadvantages of the different models. RESULTS/CONCLUSION: There are many lung cell culture models but none of these models seems to be perfect. However, they might be a great tool to perform basic research or toxicity tests. The focus here is on 3D and co-culture models, which seem to be more realistic than monocultures.