1. Izolacija in priprava celičnih linij ledvic z namenom razvoja novih in vitro modelov nefrotoksičnostiTadej Petreski, 2025, doktorska disertacija Opis: Ledvice so ključne za številne homeostatske mehanizme in so odgovorne za velik delež izločanja snovi iz telesa, zaradi česar so posebej dovzetne za delovanje nefrotoksičnih spojin. Med različnimi vrstami ledvičnih celic so proksimalne tubulne epitelijske celice (PTEC), ki sodelujejo pri tubularni sekreciji snovi, najbolj izpostavljene in zato ključnega pomena za razumevanje nefrotoksičnosti ter interakcij med zdravili v predkliničnih raziskavah. Omejena razpoložljivost in funkcionalna variabilnost komercialno dostopnih PTEC predstavlja ozko grlo pri teh raziskavah, zato je razvoj zanesljivih protokolov za njihovo izolacijo ključna za nadaljnji razvoj področja.
V tej disertaciji sta predstavljena dva optimizirana protokola za izolacijo primarnih PTEC iz rutinskih biopsij ledvičnega tkiva, pridobljenih od 13 bolnikov. Rezultat tega sta dve celični liniji, poimenovani MFUM-RPTEC-1 in MFUM-RPTEC-2. Obe celični liniji izražata za PTEC celice ključne označevalce, vključno s SGLT2, MRP4, OAT1, OAT3, OCT2, P-gp, MATE1, N-kadherinom, ZO-1 in CK-18. Ob gojenju na Transwell ploščah so celice pokazale ustrezno polarizacijo, saj so bili transportni proteini lokalizirani na predvideni (apikalni ali bazolateralni) strani, kar je bilo potrjeno tudi s konfokalno mikroskopijo.
Potencial ustvarjenih celičnih linij za uporabo v predkliničnih raziskavah smo preverjali s pripravo enostavnega in vitro modela za raziskovanje nefrotoksičnosti in interakcij med izbranimi zdravili. Celice so ohranile svojo viabilnost tudi po izpostavitvi nefrotoksičnim spojinam, kot so ciklosporin, omeprazol in njuni kombinaciji, kar dokazuje potencial celičnih linij za nadaljnji razvoj orodij in vzpostavitev raziskav na področju farmakologije ledvic.
Ključne besede: proksimalne tubulne epitelijske celice, ledvična biopsija, izolacija celic, nefrotoksičnost, in vitro model
Objavljeno v DKUM: 05.11.2025; Ogledov: 0; Prenosov: 4
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2. Vpliv dodane kokosove vode na razraščanje malinjakov v tkivni kulturi : diplomsko deloZala Orter, 2025, diplomsko delo Opis: Poskus je potekal na Fakulteti za kmetijstvo in biosistemske vede Univerze v Mariboru v obdobju od maja do junija 2025. V raziskavo smo vključili tri različna gojišča: G₁ (kontrolno gojišče brez dodatka rastnih regulatorjev), G₂ (gojišče z dodatkom 1 mg/l BAP) ter G₃ (gojišče z dodatkom 10 % kokosove vode). Skupno smo testirali 120 izsečkov malinjaka, po 40 na posamezno gojišče. Osrednji cilj raziskave je bil preučiti vpliv različnih sestav gojišč na rast in razraščanje poganjkov v in vitro pogojih, s poudarkom na primerjavi učinkovitosti rastnega regulatorja citokinina BAP in naravne sestavine kokosove vode v primerjavi s kontrolnim gojiščem brez dodanih rastnih regulatorjev. Rezultati so pokazali, da so se na kontrolnem gojišču G₁ razvili večji poganjki z dobro razvitim koreninskim sistemom, bakterijska kontaminacija pa je bila omejena predvsem na območje korenin. Gojišče G₂ je spodbudilo največje število novo tvorjenih poganjkov, bakterijska kontaminacija je bila nekoliko bolj razširjena. Gojišče G₃ je omogočilo zmeren razvoj listov, hkrati pa je bilo opaziti največjo bakterijsko kontaminacijo, kar je pripisano bogati hranilni sestavi kokosove vode, ki spodbuja rast mikroorganizmov. Ugotovljeno je bilo, da čeprav kokosova voda podpira rast rastlinskih tkiv, njena uporaba povečuje tveganje za bakterijsko kontaminacijo, kar lahko posledično oteži rast in razmnoževanje v in vitro pogojih. V okviru našega poskusa se je gojišče z dodatkom BAP izkazalo kot najučinkovitejše pri spodbujanju razmnoževanja, medtem ko uporaba kokosove vode zahteva previdno načrtovanje in optimizacijo, saj lahko zaradi svoje hranilne sestave pospeši rast nezaželenih mikroorganizmov.
Ključne besede: malinjak, kokosova voda, in vitro, tkivne kulture, Rubus idaeus
Objavljeno v DKUM: 04.09.2025; Ogledov: 0; Prenosov: 17
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3. How to establish a novel liver cell culture system that resembles the in vivo liver microenvironmentEneko Madorran, 2025, izvirni znanstveni članek Opis: Subtle physiological patterns within the human organism, such as molecular fluctuations or systemic adaptations, often remain hidden from direct observation due to the inherent variability and noise within biological samples. The liver, a vital organ essential for systemic regulation and toxicological assessment, presents this challenge due to its heightened activity, which can influence enzyme dynamics and metabolic processes. Unlike direct observation, in vitro liver models offer a more precise means of understanding these trends, providing insights into the organ's (patho)physiology, and serving as a platform for toxicity evaluation. However, current liver models lack essential features required to faithfully replicate the liver's microenvironment, resulting in reduced accuracy in toxicity assessments. Furthermore, while researchers emphasize mechanistic insights, such as the molecular processes governing glucose metabolism and cellular energy production, clinicians focus on tangible health outcomes, like blood glucose levels in patients. This disconnect between the objectives and methodologies of basic researchers and clinicians amplifies this gap, hindering effective translational research. Addressing these challenges, a novel liver cell culture system that resembles the in vivo liver microenvironment with clinical instrumentation has been proposed to enhance current liver models, improving their capacity to emulate in vivo conditions. This study introduces a novel liver cell culture system, utilizing a 96-well plate format incorporating hepatic sinusoidal endothelial cells, hepatic stellate cells, Kupffer cells, and hepatocytes to replicate the liver microenvironment. The model integrates clinical instrumentation, specifically a biochemical analyzer to ensure biomarkers closely align with those observed in clinical diagnostics. This design enables researchers to fine-tune conditions that mimic the liver's microanatomy and physiological responses, enhancing its translational potential for toxicity assessments. The approach involves primary cell culture preparation, supernatant analysis through a clinical biochemistry analyzer, and cell viability assessment using the Membrane Potential Cell Viability Assay (MPCVA) method. Additionally, advanced imaging techniques and data analysis tools are incorporated to refine the model's capabilities and ensure greater translatability to clinical applications.
Ključne besede: in vitro toxicity, liver (patho)physiology, liver in vitro model, membrane potential cell viability assay, translational research
Objavljeno v DKUM: 14.08.2025; Ogledov: 0; Prenosov: 8
Celotno besedilo (1,11 MB)
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4. The impact of temperature and the duration of freezing on a hydrogel used for a 3d-bioprinted in vitro skin modelMaja Sever, Dominik Škrinjar, Tina Maver, Monika Belak, Franc Zupanič, Ivan Anžel, Tanja Zidarič, 2024, izvirni znanstveni članek Opis: Skin bioprinting has the potential to revolutionize treatment approaches for injuries and surgical procedures, while also providing a valuable platform for assessing and screening cosmetic and pharmaceutical products. This technology offers key advantages, including flexibility and reproducibility, which enable the creation of complex, multilayered scaffolds that closely mimic the intricate microenvironment of native skin tissue. The development of an ideal hydrogel is critical for the successful bioprinting of these scaffolds with incorporated cells. In this study, we used a hydrogel formulation developed in our laboratory to fabricate a 3D-bioprinted skin model. The hydrogel composition was carefully selected based on its high compatibility with human skin cells, incorporating alginate, methyl cellulose, and nanofibrillated cellulose. One of the critical challenges in this process, particularly for its commercialization and large-scale production, is ensuring consistency with minimal batch-to-batch variations. To address this, we explored methods with which to preserve the physicochemical properties of the hydrogels, with a focus on freezing techniques. We validated the pre-frozen hydrogels’ printability, rheology, and mechanical and surface properties. Our results revealed that extended freezing times significantly reduced the viscosity of the formulations due to ice crystal formation, leading to a redistribution of the polymer chains. This reduction in viscosity resulted in a more challenging extrusion and increased macro- and microporosity of the hydrogels, as confirmed by nanoCT imaging. The increased porosity led to greater water uptake, swelling, compromised scaffold integrity, and altered degradation kinetics. The insights gained from this study lay a solid foundation for advancing the development of an in vitro skin model with promising applications in preclinical and clinical research.
Ključne besede: in vitro skin model, 3D printing, hydrogels, preclinical and clinical medicine
Objavljeno v DKUM: 28.07.2025; Ogledov: 0; Prenosov: 4
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5. Razvoj novih metod izdelave in karakterizacije mikrožilja v in vitro modelihJernej Vajda, 2025, doktorska disertacija Opis: Doktorska disertacija obravnava optimizacijo postopkov, materialov in analiznih metod v tkivnem inženirstvu mikrožilja z multidisciplinarnim pristopom, ki poleg uveljavljenih bioloških in kemijskih metod vključuje tudi računalniške simulacije, mehanske teste in nanotomografijo. V okviru raziskovalnega dela smo optimizirali linearnost ekstruzije mehanskih ekstruzijskih 3D tiskalnikov, s čimer smo dosegli boljše in bolj ponovljive rezultate 3D (bio)tiska vzorcev. Pri natisnjenih vzorcih smo primerjali uporabo CaCl2 in SrCl₂ kot ionskih zamreževalcev, pri čemer se je izkazalo, da je uporaba SrCl2 ob primerljivi celični metabolni aktivnosti izboljšala mehanske lastnosti materialov, kombinacija Sr²⁺ in Ca²⁺ pa bi lahko omogočala dodatno prilagoditev mehanskih lastnosti materialov. Uporaba Sr²⁺ hkrati zmanjša tudi vpliv kalcijevih ionov na signalizacijo celic, kar je ključno za simuliranje biološkega okolja. Na drugi strani smo s simulacijami računalniške dinamike tekočin določili mejne pogoje za razvoj peristaltične črpalke,
s katero smo eksperimentalno ovrednotili vpliv strižnih napetosti na endotelijske celice in validirali simulirane rezultate. Ločeno smo na endotelijskih celicah iz različnih virov in na podpornih celicah – kožnih fibroblastih – preiskovali lastnosti materialov in njihov vpliv na viabilnost, ohranitev celičnega fenotipa in funkcionalnosti ter celokupno metabolno aktivnost. Rezultati študij potrjujejo, da je z natančno optimizacijo procesov in materialov mogoče izboljšati funkcionalnost tkivnih in vitro modelov, kar odpira nove možnosti za napredek v tkivnem inženirstvu in regenerativni medicini.
Ključne besede: mikrožilje, in vitro model, 3D tisk, računalniška dinamika tekočin, nanotomografija
Objavljeno v DKUM: 04.07.2025; Ogledov: 0; Prenosov: 27
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6. Novel small-molecule inhibitors of the SARS-CoV-2 spike protein binding to neuropilin 1Anja Kolarič, Marko Jukič, Urban Bren, 2022, izvirni znanstveni članek Opis: Furin cleavage of the SARS-CoV-2 spike protein results in a polybasic terminal sequence
termed the C-end rule (CendR), which is responsible for the binding to neuropilin 1 (NRP1), enhancing
viral infectivity and entry into the cell. Here we report the identification of 20 small-molecule
inhibitors that emerged from a virtual screening of nearly 950,000 drug-like compounds that bind
with high probability to the CendR-binding pocket of NRP1. In a spike NRP1 binding assay, two of
these compounds displayed a stronger inhibition of spike protein binding to NRP1 than the known
NRP1 antagonist EG00229, for which the inhibition of the CendR peptide binding to NRP1 was
also experimentally confirmed. These compounds present a good starting point for the design of
small-molecule antagonists against the SARS-CoV-2 viral entry.
Ključne besede: neuropilin 1, SARS-CoV-2, COVID-19, spike binding inhibitors, virtual screening, small-molecule antagonists, molecular docking, in vitro binding assay
Objavljeno v DKUM: 09.05.2025; Ogledov: 0; Prenosov: 11
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7. Copper sulfate elicitation effect on biomass production, phenolic compounds accumulation, and antioxidant activity of Morus nigra L. stem node cultureJan Senekovič, Špela Jelen, Andreja Urbanek Krajnc, 2025, izvirni znanstveni članek Opis: Phenolic compounds are strong antioxidant and antibacterial agents with great pharmacological, medicinal, nutritional, and industrial value. The potential of Morus nigra in stem node culture was investigated for the production of phenolic compounds and their elicitation with CuSO4. Individual phenolic compounds in the samples were identified and quantified by using HPLC-PDA and HPLC-MS methods, while the content of total phenolic compounds, the content of total flavonoids, and the antioxidant activity of methanolic extracts were evaluated spectrophotometrically. The highest fresh and dry weights were obtained in plantlets treated with 0.5 mM CuSO4 for 42 days. The highest total phenolic content, total flavonoid content, and antioxidant activity of the extracts were determined in stem node cultures treated with 3 mM CuSO4 for 42 days. Under the latter conditions, the predominant representatives of the caffeoylquinic acids, p-coumaric acid derivatives, kaempferol derivatives, and quercetin derivatives also achieved the highest content. The most abundant phenolic compound in all samples was the chlorogenic acid. The nodal culture of M. nigra elicited with CuSO4 could potentially be used for the industrial production of phenolic compounds, especially caffeoylquinic acids. Moreover, considering the biochemical response to CuSO4 treatment and the ability to tolerate and accumulate copper, the potential application of M. nigra in phytoremediation is also highlighted.
Ključne besede: antioxidative potential, black mulberry, copper stress, elicitation of phenolics, flavonoids, in vitro culture, nodal culture, phenolic acids
Objavljeno v DKUM: 25.03.2025; Ogledov: 0; Prenosov: 13
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8. Comparison of conventional and supercritical isolation methods from bee pollen and their effects on the concentrations of bioactive compoundsTaja Žitek Makoter, Vesna Postružnik, Anastasia Kyriakoudi, Elli Pantazi, Natasa Kalogiouri, Ioannis Mourtzinos, Željko Knez, Maša Knez Marevci, 2025, izvirni znanstveni članek Opis: The present work represents a profound investigation of material pretreatment, comparison of different isolation methods (conventional and supercritical), and characterization of phenolic compounds and amino acids from bee pollen. The determination of amino acids was carried out after developing a novel hydrophilic interaction chromatography combined with quadrupole time-of-flight tandem mass spectrometric method (HILIC-QTOF-MS/MS) with limits of detection over the range 1 mg/kg -5 mg/kg and recoveries over the range 87.6% - 97.1%. The optimal conditions for the isolation of amino acids are in the range of temperatures between 25°C to 55°C and at pressures between 300 bar and 350 bar. It can be concluded that the extraction temperature and the pressure affect antioxidant activity. The favored conditions are lower temperatures and higher pressures.
Ključne besede: bee pollen, in vitro antioxidant activity, HILIC-LC-QTOF-MS.MS, amino acids, WM-266-4 cells
Objavljeno v DKUM: 20.03.2025; Ogledov: 0; Prenosov: 6
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9. Design of in vitro hair follicles for different applications in the treatment of alopecia : a reviewMatej Žnidarič, Žan Michel Žurga, Uroš Maver, 2021, pregledni znanstveni članek Opis: The hair research field has seen great improvement in recent decades, with in vitro hair follicle (HF) models being extensively developed. However, due to the cellular complexity and number of various molecular interactions that must be coordinated, a fully functional in vitro model of HFs remains elusive. The most common bioengineering approach to grow HFs in vitro is to manipulate their features on cellular and molecular levels, with dermal papilla cells being the main focus. In this study, we focus on providing a better understanding of HFs in general and how they behave in vitro. The first part of the review presents skin morphology with an emphasis on HFs and hair loss. The remainder of the paper evaluates cells, materials, and methods of in vitro growth of HFs. Lastly, in vitro models and assays for evaluating the effects of active compounds on alopecia and hair growth are presented, with the final emphasis on applications of in vitro HFs in hair transplantation. Since the growth of in vitro HFs is a complicated procedure, there is still a great number of unanswered questions aimed at understanding the long-term cycling of HFs without losing inductivity. Incorporating other regions of HFs that lead to the successful formation of different hair classes remains a difficult challenge.
Ključne besede: hair follicle, alopecia, dermal papilla cells, hair transplantation, in vitro hair follicle models
Objavljeno v DKUM: 11.03.2025; Ogledov: 0; Prenosov: 7
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10. An overview of the current state of cell viability assessment methods using OECD classificationEneko Madorran, Miha Ambrož, Jure Knez, Monika Sobočan, 2025, pregledni znanstveni članek Opis: Over the past century, numerous methods for assessing cell viability have been
developed, and there are many different ways to categorize these methods accordingly. We
have chosen to use the Organisation for Economic Co-operation and Development (OECD)
classification due to its regulatory importance. The OECD categorizes these methods
into four groups: non-invasive cell structure damage, invasive cell structure damage, cell
growth, and cellular metabolism. Despite the variety of cell viability methods available,
they can all be categorized within these four groups, except for two novel methods based
on the cell membrane potential, which we added to the list. Each method operates on
different principles and has its own advantages and disadvantages, making it essential
for researchers to choose the method that best fits their experimental design. This review
aims to assist researchers in making this decision by describing these methods regarding
their potential use and providing direct references to the cell viability assessment methods.
Additionally, we use the OECD classification to facilitate potential regulatory use and to
highlight the need for adding a new category to their list.
Ključne besede: cell viability, cell-based methods, in vitro toxicology, OECD cell viability classification
Objavljeno v DKUM: 13.02.2025; Ogledov: 0; Prenosov: 8
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