Standardní kultivace nádorových buněčných linií ve 2D uspořádání je dobře zavedeným a finančně dostupným experimentálním mode-lem pro in vitro testování biologických účinků potenciálních protinádorových léčiv. 2D kultury však postrádají metabolické a proliferační gradienty, důležité buněčné interakce a signalizace, které jsou přítomné in vivo. 3D buněčné sféroidy zohledňují gradienty živin, kyslíku či odpadních metabolitů, důležitost interakcí mezi buňkami a extracelulární matrix a navozují tak situaci bližší reálným podmínkám. Bio-logické vlastnosti 3D sféroidů a jejich odpovědi na účinky léčiv se značně liší ve srovnání s 2D kulturami. Hodnocením protinádorových účinků potenciálních léčiv na 3D kulturách se zásadně zvyšuje šance na výběr farmakologicky relevantních struktur a snížit tak riziko neúspěchu v průběhu klinického testování.

Traditional cultivation of cancer cell lines in 2D arrangement is well established and affordable experimental model for in vitro testing of biological effects of potential anticancer drugs. However, 2D cultures lack metabolic and proliferative gradients, important cell interac-tions and signaling that are present in vivo. Within 3D spheroids the gradients of nutrients, oxygen or waste metabolites, the importance of interactions between the cells and the extracellular matrix are included, and thus 3D can better simulate in vivo tumor microenviro-ment. The biological properties of 3D spheroids and their responses to drug effects vary greatly compared to 2D cultures. The evaluation of anticancer drug effects on 3D spheroids increases the chances of selection of pharmacologically relevant structures and thus reduce clinical trial failure risk.

• Klíčová slova
• solidní nádory,
• MeSH
• biologické modely MeSH
• buněčné sféroidy * fyziologie   klasifikace   účinky léků   MeSH
• lidé MeSH
• nádorové buňky kultivované cytologie   mikrobiologie   MeSH
• nádory diagnostické zobrazování   MeSH
• preklinické hodnocení léčiv MeSH
• protinádorové látky farmakokinetika   MeSH
• techniky 3D buněčné kultury metody   MeSH
• techniky in vitro metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Východiska: Primární lidské B buňky chronické lymfocytární leukemie (CLL) podléhají při kultivaci in vitro buněčné smrti, nicméně jejich přežití lze signifikantně prodloužit kontaktem se stromálními buňkami nebo přítomností specifických solubilních faktorů. Pro účely výzkumu chování CLL buněk jsme vytvořili 3D in vitro model, ve kterém bylo simulováno vhodné mikroprostředí pro CLL buňky umožňující studium mechanizmu jejich přežívání v dlouhodobé kultivaci. Materiál a metody: Naším cílem bylo, aby struktura scaffoldu byla geometricky podobná 3D morfologii kostní dřeně, která vyplňuje trabekulární kost, aby měl 3D scaffold dostatečně velký povrch pro zachycení buněk a zároveň velkou pórovitost pro buněčnou migraci a transport živin. Dalším požadavkem byla také alespoň částečná transparentnost potřebná pro pozorování buněčného modelu pomocí optických metod. Připravili jsme 3D scaffoldy z porózního hydrogelu poly (2-hydroxyetyl metakrylát) (pHEMA), poly (2-hydroxyetyl metakrylát-co-2-aminoetyl metakrylát) p (HEMA-co-AEMA) a p (HEMA-co-AEMA) modifikovaný s často používaným adhezním peptidem Arg-Gly-Asp (RGD). Všechny hydrogelové scaffoldy byly vyrobeny ve čtyřech velikostech pórů (125, 200, 300 a 350–450 μm). Scaffoldy byly testovány pomocí HS-5 buněčné linie odvozené z lidských stromálních buněk kostní dřeně a HEK293 buněčné linie odvozené z lidských embryonálních buněk ledvin. Výsledky: Hydrogelový scaffold p (HEMA-co-AEMA) modifikovaný adhezním peptidem Arg-Gly-Asp (RGD) s velikostí pórů 350–450 μm prokázal, že je vhodným systémem pro 3D kultivace buněk, neboť podporuje interakce mezi buňkami navzájem a také mezi buňkami a materiálem. Tento scaffold byl použit pro nasazení kultivace složené z HS-5 buněk a CLL buněk, které byly stimulovány pomocí ligandu CD40 a cytokinu IL-4. Viabilita CLL buněk byla vyšší v přítomnosti obou stimulátorů zároveň než v případě každého zvlášť. Závěr: Ukázali jsme, že technologie 3D scaffoldů je velmi dobře využitelná pro modelování mikrosystémů, kde se nádorové buňky chovají jako ve svém přirozeném mikroprostředí. Klíčová slova: hematoonkologie – leukemie – hydrogel – stromální buňky

Background: Primary human B cells chronic lymphocytic leukemia undergoes apoptosis, from which they can be rescued by contact with stromal cells or by the addition of specific soluble factor, when cultured in vitro. For research purposes of the behavior of CLL cells we created 3D in vitro model in which we simulated appropriate microenvironment for CLL cells to allow study the mechanism of survival of these cells in long-term cultivation. Material and Methods: Our aim was the scaffold structure to be geometrically similar to the 3D morphology of supporting bone marrow tissue in a trabecular bone; the 3D scaffold was also designed to conform to biocompatibility, sufficiently large surface area for cell attachment, high porosity for cell migration, proliferation and transport of nutrients. Another requirement was a partial transparency for inspection of cell model with optical techniques. We prepared 3D scaffolds from porous hydrogel poly (2-hydroxyethyl methacrylate) (pHEMA), poly (2-hydroxyethyl methacrylate-co-2-aminoethyl methacrylate) p (HEMA-co-AEMA) and p (HEMA-co-AEMA) modified with frequently used cell adhesion peptide Arg-Gly-Asp (RGD). All hydrogel scaffolds were manufactured in four pore diameters (125, 200, 300 and 350–450 μm). Scaffolds were tested with human bone marrow stromal cell line HS-5 and human embryonic kidney cell line HEK293. Results: Hydrogel scaffold p (HEMA-co-AEMA) modified with adhesion peptide Arg-Gly-Asp (RGD) with pore diameter of 350–450 μm demonstrated that it is a convenient system for 3D cell cultivation, since it promotes interaction between the cells and also between the cells and the material. This scaffold was used for seeding of co-cultivation system of HS-5 cells with CLL-cells, which were stimulated through the CD40L signaling pathway as well as via the IL-4 pathway. Viability of B-CLL cells was higher in the presence of both stimulators than with each alone. Conclusions: We have shown that 3D scaffold technology is very useful for modeling of microsystems where the cancer cells behave like in their natural microenvironment. Key words: hematooncology – leukemia – hydrogel – stromal cells This work was supported by grant COST CZ LD15144 “Cellular and acellular grounds for regeneration of bones and teeth” awarded by the Ministry of Education, Youth and Sport of the Czech Republic. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 6. 3. 2017 Accepted: 26. 3. 2017

• MeSH
• biokompatibilní materiály MeSH
• biologické modely MeSH
• chronická lymfatická leukemie patologie   MeSH
• hydrogely * MeSH
• kultivační techniky metody   MeSH
• mezenchymální kmenové buňky MeSH
• nádorové buňky kultivované * MeSH
• nádorové mikroprostředí MeSH
• techniky in vitro MeSH
• tkáňové podpůrné struktury * MeSH
• Publikační typ
• práce podpořená grantem MeSH

Studium proteinů a zvláště jejich 3D struktury či protein -proteinových interakcí hraje v dnešním biochemickém výzkumu nezanedbatelnou roli. Řada alternativních metod tohoto výzkumu (např. PIXL, FRET) využívá biotechnologické postupy pro zavedení nepřirozených aminokyselin či jejich strukturních analogů do proteinové sekvence během jejich rekombinantní přípravy. Předkládaná práce uvádí několik biotechnologických přístupů inkorporace foto -methioninu (pMet, L-2-amino-5,5-azi -hexanová kyselina) do sekvence dvou modelových savčích proteinů.

The study of proteins and especially their 3D structure or protein -protein interactions plays significant role in contemporary biochemical research. Many alternative methods of the research (e.g. PIXL, FRET) employing different biotechnology techniques to introduce the non -natural amino acids or their structural analogues within protein sequence during its recombinant expression. This study presents several biotechnology approaches to introduce photo -methionine (pMet, L-2-amino-5,5-azi -hexanoic acid) into the sequence of two model mammalian proteins.

• Klíčová slova
• světlem iniciované síťování, foto-methionin,
• MeSH
• aminoacyl-tRNA-synthetasy metabolismus   MeSH
• aminokyseliny chemie   metabolismus   MeSH
• biotechnologie MeSH
• diazomethan MeSH
• konformace proteinů * MeSH
• mapování interakce mezi proteiny * MeSH
• methionin MeSH
• posttranslační úpravy proteinů * MeSH
• proteiny chemie   MeSH
• proteosyntéza MeSH
• techniky in vitro MeSH
• zobrazování trojrozměrné MeSH
• Publikační typ
• práce podpořená grantem MeSH

Consensus toxicity factors (CTFs) were developed as a novel approach to establish toxicity factors for risk assessment of dioxin-like compounds (DLCs). Eighteen polychlorinated dibenzo-p-dioxins, dibenzofurans (PCDD/Fs), and biphenyls (PCBs) with assigned World Health Organization toxic equivalency factors (WHO-TEFs) and two additional PCBs were screened in 17 human and rodent bioassays to assess their induction of aryl hydrocarbon receptor-related responses. For each bioassay and compound, relative effect potency values (REPs) compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin were calculated and analyzed. The responses in the human and rodent cell bioassays generally differed. Most notably, the human cell models responded only weakly to PCBs, with 3,3',4,4',5-pentachlorobiphenyl (PCB126) being the only PCB that frequently evoked sufficiently strong responses in human cells to permit us to calculate REP values. Calculated REPs for PCB126 were more than 30 times lower than the WHO-TEF value for PCB126. CTFs were calculated using score and loading vectors from a principal component analysis to establish the ranking of the compounds and, by rescaling, also to provide numerical differences between the different congeners corresponding to the TEF scheme. The CTFs were based on rat and human bioassay data and indicated a significant deviation for PCBs but also for certain PCDD/Fs from the WHO-TEF values. The human CTFs for 2,3,4,7,8-pentachlorodibenzofuran, 1,2,3,4,7,8-hexachlorodibenzofuran, 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin, and 1,2,3,4,7,8,9-heptachlorodibenzofuran were up to 10 times greater than their WHO-TEF values. Quantitative structure-activity relationship models were used to predict CTFs for untested WHO-TEF compounds, suggesting that the WHO-TEF value for 1,2,3,7,8-pentachlorodibenzofuran could be underestimated by an order of magnitude for both human and rodent models. Our results indicate that the CTF approach provides a powerful tool for condensing data from batteries of screening tests using compounds with similar mechanisms of action, which can be used to improve risk assessment of DLCs.

• MeSH
• benzofurany chemie   toxicita   MeSH
• hlodavci MeSH
• krysa rodu rattus MeSH
• kvantitativní vztahy mezi strukturou a aktivitou MeSH
• lidé MeSH
• počítačová simulace MeSH
• polychlorované bifenyly chemie   toxicita   MeSH
• polychlorované dibenzodioxiny analogy a deriváty   chemie   toxicita   MeSH
• receptory aromatických uhlovodíků fyziologie   MeSH
• techniky in vitro MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Following advancements in the field of genotoxicology, it has become widely accepted that 3D models are not only more physiologically relevant but also have the capacity to elucidate more complex biological processes that standard 2D monocultures are unable to. Whilst 3D liver models have been developed to evaluate the short-term genotoxicity of chemicals, the aim of this study was to develop a 3D model that could be used with the regulatory accepted in vitro micronucleus (MN) following low-dose, longer-term (5 days) exposure to engineered nanomaterials (ENMs). A comparison study was carried out between advanced models generated from two commonly used liver cell lines, namely HepaRG and HepG2, in spheroid format. While both spheroid systems displayed good liver functionality and viability over 14 days, the HepaRG spheroids lacked the capacity to actively proliferate and, therefore, were considered unsuitable for use with the MN assay. This study further demonstrated the efficacy of the in vitro 3D HepG2 model to be used for short-term (24 h) exposures to genotoxic chemicals, aflatoxin B1 (AFB1) and methyl-methanesulfonate (MMS). The 3D HepG2 liver spheroids were shown to be more sensitive to DNA damage induced by AFB1 and MMS when compared to the HepG2 2D monoculture. This 3D model was further developed to allow for longer-term (5 day) ENM exposure. Four days after seeding, HepG2 spheroids were exposed to Zinc Oxide ENM (0-2 µg/ml) for 5 days and assessed using both the cytokinesis-block MN (CBMN) version of the MN assay and the mononuclear MN assay. Following a 5-day exposure, differences in MN frequency were observed between the CBMN and mononuclear MN assay, demonstrating that DNA damage induced within the first few cell cycles is distributed across the mononucleated cell population. Together, this study demonstrates the necessity to adapt the MN assay accordingly, to allow for the accurate assessment of genotoxicity following longer-term, low-dose ENM exposure.

• MeSH
• aflatoxin B1 toxicita   MeSH
• biologické modely MeSH
• buněčné kultury metody   MeSH
• buněčné linie MeSH
• buněčné sféroidy * MeSH
• buňky Hep G2 MeSH
• hepatocyty účinky léků   MeSH
• játra účinky léků   MeSH
• lidé MeSH
• methylmethansulfonát toxicita   MeSH
• mikrojaderné testy metody   MeSH
• mutageny toxicita   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Publikační typ
• abstrakt z konference MeSH

The testis is a priority organ for developing alternative models to assess male reproductive health hazards of chemicals. This study characterized a 3D in vitro model of murine prepubertal Leydig TM3 cells with improved expression of steroidogenesis markers suitable for image-based screening of testicular toxicity. This 3D scaffold-free spheroid model was applied to explore the impact of prototypical endocrine-disrupting chemicals (EDCs) and environmental reprotoxicants (benzo[a]pyrene, 2- and 9-methylanthracenes, fluoranthene, triclosan, triclocarban, methoxychlor) on male reproductive health. The results were compared to the male reprotoxicity potential of EDCs assessed in a traditional monolayer (2D) culture. The testicular toxicity was dependent not only on the type of culture (2D vs. 3D models) but also on the duration of exposure. Benzo[a]pyrene and triclocarban were the most active compounds, eliciting cytotoxic effects in prepubertal Leydig cells at low micromolar concentrations, which might be a mechanism contributing to their male reprotoxicity.

• MeSH
• benzopyren toxicita   MeSH
• endokrinní disruptory * chemie   MeSH
• Leydigovy buňky * MeSH
• myši MeSH
• rozmnožování MeSH
• testis MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

3D cell culture is an invaluable tool in developmental, cell, and cancer biology. By mimicking crucial features of in vivo environment, including cell-cell and cell-extracellular matrix interactions, 3D cell culture enables proper structural architecture and differentiated function of normal tissues or tumors in vitro. Thereby 3D cell culture realistically models in vivo tissue conditions and processes, and provides in vivo like responses. Since its early days in the 1970s, 3D cell culture has revealed important insights into mechanisms of tissue homeostasis and cancer, and accelerated translational research in cancer biology and tissue engineering.

• MeSH
• biomimetika MeSH
• bioprinting MeSH
• buněčné kultury metody   MeSH
• lidé MeSH
• tkáňové inženýrství MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Long-term treatment of cancer with chemotherapeutics leads to the development of resistant forms that reduce treatment options. The main associated mechanism is the overexpression of transport proteins, particularly P-glycoprotein (P-gp, ABCB1). In this study, we have tested the anticancer and multidrug resistance (MDR) modulation activity of 15 selenocompounds. Out of the tested compounds, K3, K4, and K7 achieved the highest sensitization rate in ovarian carcinoma cells (HOC/ADR) that are resistant to the action of the Adriamycin. These compounds induced oxidation stress, inhibited P-gp transport activity and altered ABC gene expression. To verify the effect of compounds, 3D cell models were used to better mimic in vivo conditions. K4 and K7 triggered the most significant ROS release. All selected selenoesters inhibited P-gp efflux in a dose-dependent manner while simultaneously altering the expression of the ABC genes, especially P-gp in paclitaxel-resistant breast carcinoma cells (MCF-7/PAX). K4, and K7 demonstrated sensitization potential in resistant ovarian spheroids. Additionally, all selected selenoesters achieved a high cytotoxic effect in 3D breast and ovarian models, which was comparable to that in 2D cultures. K7 was the only non-competitive P-gp inhibitor, and therefore appears to have considerable potential for the treatment of drug-resistant cancer.

• MeSH
• chemorezistence MeSH
• doxorubicin farmakologie   MeSH
• ketony farmakologie   MeSH
• lidé MeSH
• mnohočetná léková rezistence MeSH
• nádorové buněčné linie MeSH
• nádory prsu * farmakoterapie   MeSH
• protinádorové látky * farmakologie   terapeutické užití   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

OBJECTIVES: The purpose of this study was to investigate the effect of milk and fluoridated milk on bacterially induced caries-like lesions. SAMPLE AND METHODS: Extracted impacted human molars were cut in half and covered with a varnish leaving a 4*4 mm window. The samples were coated with biofilm of S. sobrinus and were further divided into three experimental groups of S. sobrinus, S. sobrinus and milk and S. sobrinus and fluoridated milk. As negative controls served teeth incubated in saline. Of twenty tooth halves serial ground sections were cut through the lesions and investigated with polarization light microscopy (PLM) and scanning electron microscopy (SEM) and EDX element analysis. The PLM photographs were used for 3D reconstruction, volumetric assessment and determination of the extension of the lesion zones. Of eight tooth halves the biofilm on the enamel surface was studied with SEM and EDX element analysis. RESULTS: Volumetric assessment showed a statistically significant difference in the volume of the body of the lesion and the translucent zone between the milk group and fluoridated milk group. Quantitative element analysis demonstrated significant differences between sound enamel and the superficial layer in the fluoridated milk group. The biofilm on the enamel surface showed an increased Ca content in the milk group and fluoridated milk group. CONCLUSIONS: Milk as a common nutrient seems to play a complex role in in-vitro biofilm--enamel interactions stimulating bacterial demineralization on one hand, and, as effective fluoride carrier, inhibits caries-like demineralization.

• MeSH
• biofilmy MeSH
• fluoridy farmakologie   MeSH
• lidé MeSH
• mléko chemie   MeSH
• moláry MeSH
• remineralizace zubů MeSH
• Streptococcus sobrinus MeSH
• techniky in vitro MeSH
• zubní kaz mikrobiologie   prevence a kontrola   MeSH
• zubní sklovina účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH