A Human Cell-based Assay to Assess the Induction of Vasculature Formation for Non-genotoxic Carcinogenicity Testing Purposes : A Pilot Study

The induction of vasculature formation is proposed to be a significant mechanism behind the non-genotoxic carcinogenicity of a chemical. The vasculature formation model used in this study is based on the coculture of human primary HUVECs and hASCs. This model was used to develop an assay to assess the induction of vasculature formation. Three assay protocols, based on different conditions, were developed and compared in order to identify the optimal conditions required. Some serum supplements and growth factors were observed to be essential for initiating vasculature formation. Of the studied putative positive reference chemicals, aspartame, sodium nitrite, bisphenol A and nicotine treatment led to a clear induction of vasculature formation, but arsenic and cadmium treatment only led to a slight increase. This human cell-based assay has the potential to be used as one test within a next generation testing battery, to assess the non-genotoxic carcinogenicity of a chemical through the mechanism of vasculature formation induction.publishedVersionPeer reviewe

Association between [Ga-68]NODAGA-RGDyK uptake and dynamics of angiogenesis in a human cell-based 3D model

Radiolabeled RGD peptides targeting expression of alpha(v)beta(3) integrin have been applied to in vivo imaging of angiogenesis. However, there is a need for more information on the quantitative relationships between RGD peptide uptake and the dynamics of angiogenesis. In this study, we sought to measure the binding of [Ga-68]NODAGA-RGDyK to alpha(v)beta(3) integrin in a human cell-based three-dimensional (3D) in vitro model of angiogenesis, and to compare the level of binding with the amount of angiogenesis. Experiments were conducted using a human cell-based 3D model of angiogenesis consisting of co-culture of human adipose stem cells (hASCs) and of human umbilical vein endothelial cells (HUVECs). Angiogenesis was induced with four concentrations (25%, 50%, 75%, and 100%) of growth factor cocktail resulting in a gradual increase in the density of the tubule network. Cultures were incubated with [Ga-68]NODAGA-RGDyK for 90 min at 37 degrees C, and binding of radioactivity was measured by gamma counting and digital autoradiography. The results revealed that tracer binding increased gradually with neovasculature density. In comparison with vessels induced with a growth factor concentration of 25%, the uptake of [Ga-68]NODAGA-RGDyK was higher at concentrations of 75% and 100%, and correlated with the amount of neovasculature, as determined by visual evaluation of histological staining. Uptake of [Ga-68]NODAGA-RGDyK closely reflected the amount of angiogenesis in an in vitro 3D model of angiogenesis. These results support further evaluation of RGD-based approaches for targeted imaging of angiogenesis

Functional human cell-based vascularised cardiac tissue model for biomedical research and testing

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) are widely used in in vitro biomedical research and testing. However, fully matured, adult cardiomyocyte characteristics have not been achieved. To improve the maturity and physiological relevance of hiPSC-derived cardiomyocytes, we co-cultured them with preconstructed vascular-like networks to form a functional, human cell-based cardiac tissue model. The morphology and gene expression profiles indicated advanced maturation in the cardiac tissue model compared to those of a cardiomyocyte monoculture. The cardiac tissue model’s functionality was confirmed by measuring the effects of 32 compounds with multielectrode array and comparing results to human data. Our model predicted the cardiac effects with a predictive accuracy of 91%, sensitivity of 90% and specificity of 100%. The correlation between the effective concentration (EC50) and the reported clinical plasma concentrations was 0.952 (R2 = 0.905). The developed advanced human cell-based cardiac tissue model showed characteristics and functionality of human cardiac tissue enabling accurate transferability of gained in vitro data to human settings. The model is standardized and thus, it would be highly useful in biomedical research and cardiotoxicity testing.publishedVersionPeer reviewe

Evaluation of Neurotoxicity of Mercury Compounds and Aluminum in Cell Cultures

Elohopea ja alumiini ympäristömyrkkyjä, jotka ovat haitallisia aivojen toiminnalle. Pitkäaikainen altistus näille metalleille voi johtaa jopa hermosolujen tuhoutumiseen ja hermoston rappeutumissairauksiin. Ihmisten altistuminen on yleistä, sillä ympäristön saastumisen ja teollisen hyödyntämisen takia elohopeaa ja alumiinia on aina ympäristössä läsnä. Aivot on suojattu suhteellisen hyvin haitallisia kemikaaleja vastaan, mutta alumiini ja metyylielohopea pystyvät ylittämään ns. veriaivoesteen ja kerääntymään aivoihin. Ne haittaavat monia solutasoisia aineenvaihduntatoimintoja, sillä ne matkivat ja korvaavat solujen käyttämiä luonnollisia metalleja ja voivat sitoutua solun rakenneosasten kanssa. Vaikutukset kohdistuvat hyvinkin erilaisiin kohteisiin soluissa. Soluviljelmissä tutkimusolosuhteet ovat hallittavissa, ja spesifien vaikutusten arviointi eri menetelmien on mahdollista. Väitöskirjatyössä tutkittiin aivoperäisissä soluviljelmissä epäorgaanisen ja orgaanisen elohopean ja alumiinin vaikutuksia. Tutkimuskohteina olivat yleinen solumyrkyllisyys ja solukuoleman mekanismit, sekä tarkemmat solutasoiset vaikutukset. Tällaisia olivat aivojen tärkeimmän välittäjäaineen, glutamaatin, sisäänottoon liittyvät reaktiopolut, solunsisäisen kalsiumin määrä ja aivojen tukisolujen, gliasolujen, aktivaatio. Aineiden läpäisevyys veriaivoesteen läpi määrää paljolti aineen mahdollisen aivomyrkyllisyyden. Työssä kehiteltiin veriaivoesteen soluviljelymallia, jossa testattiin elohopean ja alumiinin kulkua esteen läpi. Tulokset osoittivat, että tutkittavista aineista metyylielohopea oli haitallisin kaikissa solumalleissa jo pienillä pitoisuuksilla. Lisäksi metyylielohopean vaikutus ei ollut palautuva altistuksen loputtua, kuten epäorgaanisen elohopean ja alumiinin. Alumiini ei aiheuttanut solukuolemia pienillä pitoisuuksilla, mutta merkittävä entsyymien aktivaatio oli odottamaton löydös. Aktivaatiolla voi olla merkitystä erityisesti alumiinin haittavaikutusten alkamisvaiheessa. Tutkitut metallit pystyivät aiheuttamaan soluviljelmissä hermoston tukisolujen aktivaation, joka on yleinen merkki soluvaurioista. Tukisolut ylläpitävät ihanteellista ympäristöä hermosoluille, joten tukisolujen vauriot vaikuttavat suuresti hermosoluihin. Elohopea esti glutamaatin sisäänottoa soluihin, mistä voi olla vakavia seurauksia ympäröiville soluille. Työssä kehitetty kolmen eri solutyypin veriaivoestemalli pystyi erottamaan eri elohopeatyyppien ja alumiin myrkyllisyyden. Tällainen erityisesti ihmissoluista rakennettu veriaivoestemalli on arvokas uusien kemikaalien testaamisessa. Tutkimus osoitti useita haittavaikutuksia hermostoperäisissä soluissa pitoisuuksissa, jotka ovat mahdollisia myös ihmisaltistuksissa. Metyylielohopean myrkyllisyys korostui myös siksi, että elohopea esiintyy luonnossa yleensä metyylielohopeamuodossa, joka pääsee helposti aivoihin. Alumiinin solumyrkyllisyys oli vähäisempää, mutta alumiinin aiheuttamat erityiset vaikutukset voivat olla yhtä merkittäviä ja välittää solutuhoja.Mercury and aluminum are neurotoxic metals with diverse effects on cellular functions in the brain. Ultimately exposure to them can lead to neural destruction and degenerative diseases. Although their toxic potency is now widely known, their existence in the environment and in several man-made applications makes human exposure inevitable. There are many mechanisms that cause cellular destruction with a delicate interplay with each other. That is why studies on different adverse mechanisms, and new methodological developments, as applied in this work, broaden the knowledge of the toxicity of these metals. Cell culture systems make such studies possible in strictly defined conditions. For the experiments on mercuric mercury, methylmercury and aluminum toxicity, several methods and cultures of different neural cell types were used. Cytotoxicity was evaluated in neuroblastoma, glioblastoma and retinal pigment epithelial (RPE) cell lines, with a method based on measuring the mitochondrial integrity, WST-1 assay, and the leakage of lactate dehydrogenase enzyme (LDH-test). To further characterise the mechanism of cell deaths in these experiments, induction of apoptosis, the cellular self-destruction process, was evaluated. The reactivity of glial primary astrocytes as a response to toxic insults was evaluated by measuring the amount of an intermediary filament protein, the glial fibrillary acidic protein (GFAP). The uptake of the main excitatory amino acid glutamate was studied in connection with mercury in primary RPE cells. Evaluations of protein kinase C (PKC)-linked pathways and intracellular calcium level were included to characterise the effect. For assessing how well the study metals can pass the blood-brain barrier (BBB), an in vitro BBB barrier model built on transparent membrane filters was established. Furthermore, cellular morphology was one of the aspects monitored throughout the study. The cytotoxicity studies showed that methylmercury was the most toxic substance in the sense that it exerted its effects at lower concentrations than either mercuric mercury or aluminum. The effect was seen in all culture systems. Apoptotic cell death mechanisms were involved with all metals studied, but with different cell specificity. An unexpected finding was the activation of mitochondrial dehydrogenases, especially in connection with methylmercury and aluminum at low concentrations. The activation as a toxic response may lead to equally significant consequences as deactivations seen in cytotoxicity studies. An important result was that methylmercury toxicity seemed to be irreversible. Aluminum was not very cytotoxic (did not cause cell deaths), but showed responses that may be equally important, e.g. mitochondrial activation. Formation of fibrillary structures characteristic of aluminum exposure was especially seen in glial cells, not so much in neuronal cells as had been usual in previous studies. The induction of GFAP synthesis was adapted for in vitro system. The GFAP synthesis was induced with exposure to all the metals studied. The cellular structural filaments may be a sensitive target common to many toxic metals, since mercurial compounds were as active inducers of GFAP production as aluminum. Mercuric mercury inhibited glutamate uptake in RPE cells. The inhibition was not permanent, since the uptake could mostly be restored by activating the PKC. When glutamate accumulates in the extracellular space of RPE cells, excitotoxic damages in neighbouring neuronal cells may follow. Also another major mechanism that mediates mercury toxicity was shown: Mercuric mercury could increase the intracellular calcium level rapidly from the extracellular calcium pools. Calcium is connected to several toxic cellular reactions. One of the main outcomes was that an in vitro BBB model constructed from human cells was developed. The model was able to distinguish the toxicity differences of methylmercury, mercuric mercury and aluminum. The in vitro BBB model can be adapted for the testing of new chemicals and drugs for their potency to cross the BBB and therefore exert adverse effects in the brain. As a conclusion, mercuric mercury, methylmercury and aluminum showed diverse adverse effects on neural cells. Due to accumulation, the effective concentrations may be exceeded even in human exposures. The study especially emphasised the toxicity of methylmercury, because of its wide potency and irreversibility of the effects. Furthermore, methylmercury is the form of mercury that easily enters the brain. Aluminum seemed to be less cytotoxic, but the specific effects induced by aluminum may initiate or mediate the cellular destruction as well

Evaluation of Neurotoxicity of Mercury Compounds and Aluminum in Cell Cultures

Elohopea ja alumiini ympäristömyrkkyjä, jotka ovat haitallisia aivojen toiminnalle. Pitkäaikainen altistus näille metalleille voi johtaa jopa hermosolujen tuhoutumiseen ja hermoston rappeutumissairauksiin. Ihmisten altistuminen on yleistä, sillä ympäristön saastumisen ja teollisen hyödyntämisen takia elohopeaa ja alumiinia on aina ympäristössä läsnä. Aivot on suojattu suhteellisen hyvin haitallisia kemikaaleja vastaan, mutta alumiini ja metyylielohopea pystyvät ylittämään ns. veriaivoesteen ja kerääntymään aivoihin. Ne haittaavat monia solutasoisia aineenvaihduntatoimintoja, sillä ne matkivat ja korvaavat solujen käyttämiä luonnollisia metalleja ja voivat sitoutua solun rakenneosasten kanssa. Vaikutukset kohdistuvat hyvinkin erilaisiin kohteisiin soluissa. Soluviljelmissä tutkimusolosuhteet ovat hallittavissa, ja spesifien vaikutusten arviointi eri menetelmien on mahdollista. Väitöskirjatyössä tutkittiin aivoperäisissä soluviljelmissä epäorgaanisen ja orgaanisen elohopean ja alumiinin vaikutuksia. Tutkimuskohteina olivat yleinen solumyrkyllisyys ja solukuoleman mekanismit, sekä tarkemmat solutasoiset vaikutukset. Tällaisia olivat aivojen tärkeimmän välittäjäaineen, glutamaatin, sisäänottoon liittyvät reaktiopolut, solunsisäisen kalsiumin määrä ja aivojen tukisolujen, gliasolujen, aktivaatio. Aineiden läpäisevyys veriaivoesteen läpi määrää paljolti aineen mahdollisen aivomyrkyllisyyden. Työssä kehiteltiin veriaivoesteen soluviljelymallia, jossa testattiin elohopean ja alumiinin kulkua esteen läpi. Tulokset osoittivat, että tutkittavista aineista metyylielohopea oli haitallisin kaikissa solumalleissa jo pienillä pitoisuuksilla. Lisäksi metyylielohopean vaikutus ei ollut palautuva altistuksen loputtua, kuten epäorgaanisen elohopean ja alumiinin. Alumiini ei aiheuttanut solukuolemia pienillä pitoisuuksilla, mutta merkittävä entsyymien aktivaatio oli odottamaton löydös. Aktivaatiolla voi olla merkitystä erityisesti alumiinin haittavaikutusten alkamisvaiheessa. Tutkitut metallit pystyivät aiheuttamaan soluviljelmissä hermoston tukisolujen aktivaation, joka on yleinen merkki soluvaurioista. Tukisolut ylläpitävät ihanteellista ympäristöä hermosoluille, joten tukisolujen vauriot vaikuttavat suuresti hermosoluihin. Elohopea esti glutamaatin sisäänottoa soluihin, mistä voi olla vakavia seurauksia ympäröiville soluille. Työssä kehitetty kolmen eri solutyypin veriaivoestemalli pystyi erottamaan eri elohopeatyyppien ja alumiin myrkyllisyyden. Tällainen erityisesti ihmissoluista rakennettu veriaivoestemalli on arvokas uusien kemikaalien testaamisessa. Tutkimus osoitti useita haittavaikutuksia hermostoperäisissä soluissa pitoisuuksissa, jotka ovat mahdollisia myös ihmisaltistuksissa. Metyylielohopean myrkyllisyys korostui myös siksi, että elohopea esiintyy luonnossa yleensä metyylielohopeamuodossa, joka pääsee helposti aivoihin. Alumiinin solumyrkyllisyys oli vähäisempää, mutta alumiinin aiheuttamat erityiset vaikutukset voivat olla yhtä merkittäviä ja välittää solutuhoja.Mercury and aluminum are neurotoxic metals with diverse effects on cellular functions in the brain. Ultimately exposure to them can lead to neural destruction and degenerative diseases. Although their toxic potency is now widely known, their existence in the environment and in several man-made applications makes human exposure inevitable. There are many mechanisms that cause cellular destruction with a delicate interplay with each other. That is why studies on different adverse mechanisms, and new methodological developments, as applied in this work, broaden the knowledge of the toxicity of these metals. Cell culture systems make such studies possible in strictly defined conditions. For the experiments on mercuric mercury, methylmercury and aluminum toxicity, several methods and cultures of different neural cell types were used. Cytotoxicity was evaluated in neuroblastoma, glioblastoma and retinal pigment epithelial (RPE) cell lines, with a method based on measuring the mitochondrial integrity, WST-1 assay, and the leakage of lactate dehydrogenase enzyme (LDH-test). To further characterise the mechanism of cell deaths in these experiments, induction of apoptosis, the cellular self-destruction process, was evaluated. The reactivity of glial primary astrocytes as a response to toxic insults was evaluated by measuring the amount of an intermediary filament protein, the glial fibrillary acidic protein (GFAP). The uptake of the main excitatory amino acid glutamate was studied in connection with mercury in primary RPE cells. Evaluations of protein kinase C (PKC)-linked pathways and intracellular calcium level were included to characterise the effect. For assessing how well the study metals can pass the blood-brain barrier (BBB), an in vitro BBB barrier model built on transparent membrane filters was established. Furthermore, cellular morphology was one of the aspects monitored throughout the study. The cytotoxicity studies showed that methylmercury was the most toxic substance in the sense that it exerted its effects at lower concentrations than either mercuric mercury or aluminum. The effect was seen in all culture systems. Apoptotic cell death mechanisms were involved with all metals studied, but with different cell specificity. An unexpected finding was the activation of mitochondrial dehydrogenases, especially in connection with methylmercury and aluminum at low concentrations. The activation as a toxic response may lead to equally significant consequences as deactivations seen in cytotoxicity studies. An important result was that methylmercury toxicity seemed to be irreversible. Aluminum was not very cytotoxic (did not cause cell deaths), but showed responses that may be equally important, e.g. mitochondrial activation. Formation of fibrillary structures characteristic of aluminum exposure was especially seen in glial cells, not so much in neuronal cells as had been usual in previous studies. The induction of GFAP synthesis was adapted for in vitro system. The GFAP synthesis was induced with exposure to all the metals studied. The cellular structural filaments may be a sensitive target common to many toxic metals, since mercurial compounds were as active inducers of GFAP production as aluminum. Mercuric mercury inhibited glutamate uptake in RPE cells. The inhibition was not permanent, since the uptake could mostly be restored by activating the PKC. When glutamate accumulates in the extracellular space of RPE cells, excitotoxic damages in neighbouring neuronal cells may follow. Also another major mechanism that mediates mercury toxicity was shown: Mercuric mercury could increase the intracellular calcium level rapidly from the extracellular calcium pools. Calcium is connected to several toxic cellular reactions. One of the main outcomes was that an in vitro BBB model constructed from human cells was developed. The model was able to distinguish the toxicity differences of methylmercury, mercuric mercury and aluminum. The in vitro BBB model can be adapted for the testing of new chemicals and drugs for their potency to cross the BBB and therefore exert adverse effects in the brain. As a conclusion, mercuric mercury, methylmercury and aluminum showed diverse adverse effects on neural cells. Due to accumulation, the effective concentrations may be exceeded even in human exposures. The study especially emphasised the toxicity of methylmercury, because of its wide potency and irreversibility of the effects. Furthermore, methylmercury is the form of mercury that easily enters the brain. Aluminum seemed to be less cytotoxic, but the specific effects induced by aluminum may initiate or mediate the cellular destruction as well

Direct Contraction Force Measurements of Engineered Cardiac Tissue Constructs With Inotropic Drug Exposure

Contractility is one of the most crucial functions of the heart because it is directly related to the maintenance of blood perfusion throughout the body. Both increase and decrease in contractility may cause fatal consequences. Therefore, drug discovery would benefit greatly from reliable testing of candidate molecule effects on contractility capacity. In this study, we further developed a dual-axis piezoelectric force sensor together with our human cell–based vascularized cardiac tissue constructs for cardiac contraction force measurements. The capability to detect drug-induced inotropic effects was tested with a set of known positive and negative inotropic compounds of isoprenaline, milrinone, omecamtiv mecarbil, propranolol, or verapamil in different concentrations. Both positive and negative inotropic effects were measurable, showing that our cardiac contraction force measurement system including a piezoelectric cantilever sensor and a human cell–based cardiac tissue constructs has the potential to be used for testing of inotropic drug effects.publishedVersionPeer reviewe

Supplemental Material - A Human Cell-based Assay to Assess the Induction of Vasculature Formation for Non-genotoxic Carcinogenicity Testing Purposes: A Pilot Study

Supplemental Material for A Human Cell-based Assay to Assess the Induction of Vasculature Formation for Non-genotoxic Carcinogenicity Testing Purposes: A Pilot Study by Veera Hautanen, Tarja Toimela, Martin Paparella, and Tuula Heinonen in Alternatives to Laboratory Animals</p

Cytotoxicity of Water Samples Condensed from Indoor Air : An Indicator of Poor Indoor Air Quality

Introduction: The impurities of the air inside the buildings and the resulting adverse health effects have become an increasing problem. Typically indoor air impurities are mixtures of many chemical substances at relative low concentrations. The toxicity of individual substances may be negligible, the mixture effect being the primary cause for toxicity and the potential adverse health effects. Standardized screening methods for identifying the health hazard are lacking. The aim of this study was, by using conventional cytotoxicity/cell viability assays, to investigate whether indoor air cytotoxicity can be detected from water samples condensed from indoor air. Materials and Methods: The cytotoxicity of 712 water samples condensed from indoor air was investigated. First, 24 samples were tested in four different cell types (human bronchial epithelial cells, BJ fibroblasts, Tohoku Hospital Pediatric-1 [THP-1] monocytes, and THP-1 macrophages) using neutral red uptake and water-soluble tetrazolium salt 1 (WST-1) assays. Thereafter, 688 samples were tested using THP-1 macrophage/WST-1 assay. All samples were tested at 10% sample concentration, 56 samples were also tested at 25% concentration to see dose-response effects. Results: THP-1 macrophage/WST-1 assay was the most reproducible method for assessing indoor air cytotoxicity. The adverse effects of indoor air samples on THP-1 cells ranged from ca. 33% loss in cell viability to ca. 17% increase in mitochondrial activity ("cell stress"). Indoor air samples from 75% sampling sites where people reported health symptoms caused adverse effects in THP-1 macrophage/WST-1 assay. Conclusions: The assessment of indoor air cytotoxicity using water samples condensed from the indoor air and THP-1 macrophage/WST-1 assay provides a novel and practical biological approach for investigating indoor air quality. This method does not replace existing methods, but supplements them and provides a fast and cheap alternative for the first-stage screening for recognizing poor indoor air regardless of its source.publishedVersionPeer reviewe