Fabrication and characterization of microstructured scaffolds for complex 3D cell cultures

In einem natürlichen Gewebe wird das zelluläre Verhalten durch Stimuli der Mikroumgebung reguliert. Verschiedene chemische, mechanische und physikalische Reize befinden sich in einem lokalen Milieu und versorgen die Zellen mit einem biologischen Kontext. Im Vergleich zur in vivo Situation, zeigen Standard 2D in vitro Zellkulturmodelle viele Unterschiede in der zellulären Mikroumgebung und können infolgedessen eine Veränderung der Zellantwort verursachen. Die Schaffung einer physiologisch realistischeren Umgebung auf künstlichem Substrat ist ein Schlüsselfaktor für die Entwicklung zuverlässiger Plattformen, die es den kultivierten Zellen ermöglichen, sich natürlicher zu verhalten. Daher sind neuartige Substrate auf Biomaterialbasis mit maßgeschneiderten Eigenschaften sehr gefragt. Die Mikrotechnik ist ein leistungsstarkes Werkzeug, das bei der Herstellung der Funktionsgerüste hilft, um verschiedene Eigenschaften der in vivo Umgebung zu reproduzieren und auf in vitro Bedingungen zu übertragen. Die Gerüstkonstruktionsparameter können manipuliert werden, um die für das jeweilige Gewebe spezifischen Anforderungen zu erfüllen. Eine der grundlegenden Einschränkungen bei aktuellen Herstellungsverfahren ist jedoch die Unfähigkeit, mehrere Gerüsteigenschaften auf vorgefertigte Weise in eine einzelne Gerüststruktur zu integrieren. Diese Dissertation befasst sich mit Gerüstmikrofabrikations- und Oberflächenmodifikations-techniken, welche die Mikrostrukturierungstechnologie verwenden und die gleichzeitige Kontrolle über verschiedene Gerüsteigenschaften ermöglichen. Diese Ansätze bei der Mikrofabrikation von Polymergerüsten werden verwendet, um physikalische und chemische Eigenschaften bereitzustellen, die für die Leberzellkultur optimiert sind. Die physikochemischen Aspekte, die die zelluläre Mikroumgebung von Lebergewebe in vivo ausmachen, werden diskutiert und anschließend werden relevante Technologien vorgestellt, mit denen einige dieser Aspekte in vitro reguliert werden können. Im ersten Teil dieser Arbeit wird ein neuartiges zweistufiges Verfahren zur Herstellung von Polymergerüsten mit mikroporöser Struktur und definierter Topographie gezeigt. Um 3D-Matrizen mit integrierter Porosität zu erhalten, wurde nach der Herstellung mikroporöser Folien ein Mikrostrukturierungsprozess unter Verwendung der Mehrschicht Polymer-Thermoformtechnologie durchgeführt. Diese Methoden wurden verwendet, um Substrate für die organotypische 3D-Hepatozytenkultivierung herzustellen. Poröse Gerüste mit Mikrokavitäten wurden aus lösungsmittelgegossenen und phasengetrennten Polymilchsäure (PLA) Folien gebildet. Die Proben wurden auf grundlegende mechanische und Oberflächenspezifische Eigenschaften sowie auf die Zellleistung untersucht. Um einen Bezugspunkt für die Bewertung der hergestellten Matrices bereitzustellen, wurden PLA-Gerüste mit zuvor beschriebenen Substraten auf Polycarbonat (PC)-Basis mit ähnlicher Geometrie verglichen. HepG2-Zellen, die in PLA-Gerüsten kultiviert wurden, zeigten eine gewebeartige 3D-Aggregation und eine erhöhte Sekretionsrate von Albumin im Vergleich zu PC-Gerüsten. Anschließend wurde dieses zweistufige Herstellungsverfahren verwendet, um schnell abbaubare Gerüste für die gerüstfreie Zellblatttechnik herzustellen. Gerüste mit kontrollierter Porosität und Topographie, die die Schlüsselmerkmale von Lebersinusoiden nachahmen, wurden aus Poly(milch-co-glykolsäure) (PLGA)-Copolymer hergestellt und für den in vitro Abbau in Zellkultur charakterisiert. Um die Beziehung zwischen dem Abbau des Gerüsts und der Organisation der Zellen in der PLGA-Matrix aufzudecken, wurde die Lebensfähigkeit und Morphologie der kultivierten Zellen zusammen mit der Morphologie des Gerüsts untersucht. Im zweiten Teil dieser Arbeit wurden verschiedene technische Lösungen für die gerichtete Strukturierung mikroporöser Polymergerüste bewertet und ihre Eignung zur Erzeugung einer benutzerdefinierten lebenswichtigen oligozellulären Morphologie auf künstlichem Substrat vorgestellt. Besonderes Augenmerk wurde auf das 3D-Mikrokontaktdruckverfahren (3DµCP) gelegt, das die Vorteile des Mikrothermoformens und des Mikrokontaktdrucks kombiniert und eine räumlich-zeitliche Kontrolle über morphologische und chemische Merkmale in einem einzigen Schritt ermöglicht. Um das Potenzial dieser Technik aufzuzeigen, wurden Gerüste mit bestimmten Mikrostrukturen wie Kanäle mit verschiedenen Tiefen und Breiten sowie komplexere Muster hergestellt und verschiedene ECM-Moleküle gleichzeitig in die vordefinierten Geometrien übertragen. Die Gültigkeit des 3DµCP-Prozesses wurde durch mikroskopische Messungen, Fluoreszenzfärbung und Testen der Substrate auf Zelladhäsionsantwort gezeigt. Schließlich wird in dieser Arbeit die Herstellungsmethode zur Erzeugung komplexer Gerüste für die 3D- und gesteuerte Co-Kultivierung von Leberzellen vorgestellt. Polymermatrizen, die die grundlegende Leberarchitektur replizieren und somit eine gut organisierte Leberzellzusammensetzung ermöglichen, wurden erfolgreich unter Verwendung der 3DµCP-Methode hergestellt. Auf der Polycarbonatoberfläche wurden gleichzeitig chemische und topografische Leitfäden in Form sinusförmiger Strukturen strukturiert. Um die 3D-Gewebemikrostruktur zu replizieren, wurden EA.hy926- und HepG2-Zellen auf beiden Seiten des strukturierten porösen Gerüsts Co-kultiviert und anschließend einander gegenüber gestapelt, wodurch zugehörige Kanäle zur Bildung einer Kapillare führen. Das Potenzial unseres 3DµCP-strukturierten Gerüsts für die gerichtete Co-Kultivierung von Zellen wurde unter statischen Zellkulturbedingungen demonstriert. Am Ende wurden Gerüste für die weiteren Anwendungen im perfundierten Bioreaktorsystem angepasst.In a natural tissue, cellular behavior is regulated by microenvironmental stimuli. Different chemical, mechanical and physical cues reside in a local milieu and provide cells with a biological context. Compare to the in vivo situation, standard 2D in vitro cell culture models show many differences in the cellular microenvironment and as a consequence can cause alteration in cellular response. Creating physiologically more realistic environment on artificial substrate is a key factor for development of reliable platforms that enables the cultured cells to behave in a more natural manner. Therefore, novel biomaterial-based substrates with tailored properties are highly demanded. Microtechnology is a powerful tool that helps in the production of the functional scaffolds for reproducing various characteristics of the in vivo environment and transfer them to in vitro conditions. Scaffold design parameters can be manipulated to meet the needs specific to given tissue. However, one of the fundamental limitations in current fabrication methods is the inability to integrate multiple scaffold characteristics within a single scaffold structure in a pre-designed manner. This dissertation discusses scaffold microfabrication and surface modification techniques that use microstructuring technology and allows simultaneous control over various scaffold properties. These approaches in microfabricating polymeric scaffolds are used to provide physical and chemical characteristic those are more optimal for liver cell culture. The physiochemical aspects that constitute the in vivo cellular microenvironment of liver tissue are discussed and subsequently relevant technologies that can be used to regulate some of those aspects in vitro are presented. In the first part this thesis demonstrates a novel two-step procedure for manufacturing polymeric scaffolds with microporous structure and defined topography. To achieve 3D matrixes with integrated porosity, fabrication of microporous foils was followed by microstructuring process using multilayer polymer thermoforming technology. These methods were used to produce substrates for organotypic 3D hepatocyte cultivation. Porous scaffolds with the structure of microcavities were formed from solvent casted and phase separated polylactic acid (PLA) foils. Samples were investigated for basic mechanical and surface properties as well as cellular performance. Moreover, to provide a reference point for the evaluation of produced matrixes, PLA scaffolds were compared to previously reported polycarbonate (PC) based substrates with similar geometry. HepG2 cells cultured within PLA scaffolds showed 3D tissue-like aggregation and enhanced secretion rate of albumin in comparison to PC scaffolds. Subsequently, this two-step fabrication method was used to produce fast degradable scaffolds for scaffold-free cell sheet engineering. Scaffolds with controlled porosity and topography mimicking the key features of liver sinusoids were produced from poly(lactic-co-glycolic acid) (PLGA) copolymer and characterized for in vitro degradation in cell culture. To reveal the relationship between degradation of the scaffold and organization of the cells in PLGA matrix, viability and morphology of the cultured cells was examined along with scaffolds morphology. In the second part of this work, various technical solutions for directed patterning of microporous polymer scaffolds were evaluated and their suitability for creating a user-defined vital oligocellular morphology on artificial substrate was presented. Special attention was given to the 3D microcontact printing (3DµCP) method that combines the advantages of microthermoforming and microcontact printing and provides spatiotemporal control over morphological and chemical feature in a single step. To show the potential of this technique, scaffolds with determined microstructures like channels with various depths and widths as well as more complex patterns were fabricated and different ECM molecules were simultaneously transferred inside the predesigned geometries. The validity of 3DµCP process has been demonstrated by microscopic measurements, fluorescence staining and testing the substrates to cell adhesion response. Finally, this thesis presents fabrication method for manufacturing complex scaffolds for 3D and guided co-cultivation of liver cells. Polymer matrixes that replicate basic liver architecture and thus facilitate well-organized hepatic cell composition were successfully produced using the 3DµCP method. Chemical and topographical guidance cues in the form of sinusoidal structures were simultaneously patterned on the polycarbonate surface. To replicate 3D tissue microstructure, EA.hy926 and HepG2 cells were co-cultured on both sides of the patterned porous scaffold and subsequently stacked facing each other by virtue of which associated channels results in the formation of a capillary. The potential of our 3DµCP patterned scaffold for directed co-cultivation of cells was demonstrated under static cell culture conditions. At the end, scaffolds were adapted for the further applications in perfused bioreactor system

Donor-acceptor Stenhouse adduct-grafted polycarbonate surfaces: selectivity of the reaction for secondary amine on surface

Donor–acceptor Stenhouse adducts (DASAs) are gaining attention from organic and material chemists due to their visible light-stimulated photochromic properties. In this report, we present a facile method for grafting coloured triene on polycarbonate surface, without involving any pre-treatments like plasma activation, etc. The chemoselectivity of carbonate with a primary amine and Meldrum's activated furan (MAF) with polymer bound secondary amine has been exploited to graft photoswitchable DASA on the polymer surface. Primary, secondary and tertiary amine-functionalized polycarbonate surfaces have been prepared to evaluate the reactivity of amine with MAF

Long-term lipoprotein apheresis in the treatment of severe familial hypercholesterolemia refractory to high intensity statin therapy: Three year experience at a lipoprotein apheresis centre

Background: Severe familial hypercholesterolemia (FH) individuals, refractory to conventional lipidloweringmedications are at exceptionally high risk of cardiovascular events. The established therapeuticoption of last choice is lipoprotein apheresis (LA). Herein, it was sought to investigate the clinical usefulnessof LA in a highly selected group of severe heterozygous FH (HeFH), as recently described by theInternational Atherosclerosis Society (IAS), for their efficacy in lipid reduction and safety.Methods: Efficacy and safety of LA were investigated in 318 sessions of 7 severe HeFH females withcardiovascular disease, over a mean period of 26.9 ± 6.5 months. Relative reduction of low density lipoproteincholesterol (LDL-C) ≥ 60%, clinical complications and vascular access problems were evaluatedand compared between the direct adsorption of lipoproteins (DALI) and lipoprotein filtration (MembraneFiltration Optimized Novel Extracorporeal Treatment [MONET]). Additionally, lipoprotein (a)[Lp(a)], total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), triglycerides (TG) andfibrinogen concentrations were investigated.Results: The relative reduction of LDL-C, TC, TG and Lp(a) were 69.4 ± 12.9%, 59.7 ± 9.1, 51.5 ±± 14.2% and 71.3 ± 14.4%, respectively. A similar efficacy was found in both systems in LDL-C removal.DALI system led to larger depletions of Lp(a) (80.0 [76–83]% vs. 73.0 [64.7–78.8]%; p < 0.001).The frequency of clinical side effects and vascular access problems were low (8.5%).Conclusions: Long-term LA in severe HeFH individuals is safe and efficiently reduces LDL-C andLp(a). Higher efficacy of the DALI system than MONET in Lp(a) removal may indicate the need for individualizedapplication of the LA system in severe HeFH individuals

MatriGrid® based biological morphologies: tools for 3D cell culturing

Recent trends in 3D cell culturing has placed organotypic tissue models at another level. Now, not only is the microenvironment at the cynosure of this research, but rather, microscopic geometrical parameters are also decisive for mimicking a tissue model. Over the years, technologies such as micromachining, 3D printing, and hydrogels are making the foundation of this field. However, mimicking the topography of a particular tissue-relevant substrate can be achieved relatively simply with so-called template or morphology transfer techniques. Over the last 15 years, in one such research venture, we have been investigating a micro thermoforming technique as a facile tool for generating bioinspired topographies. We call them MatriGrid ® s. In this research account, we summarize our learning outcome from this technique in terms of the influence of 3D micro morphologies on different cell cultures that we have tested in our laboratory. An integral part of this research is the evolution of unavoidable aspects such as possible label-free sensing and fluidic automatization. The development in the research field is also documented in this account

Samotność idei? : społeczeństwo obywatelskie we współczesnym świecie

Publikacja recenzowana / Peer-reviewed publicationCelem niniejszej pracy jest próba przedstawienia kształtu obecnych relacji i stosunków między społeczeństwem obywatelskim a wspomnianymi ideami czy procesami społecznymi.Książka, którą oddajemy do rąk Czytelników jest zbiorem prac studialnych o zróżnicowanym poziomie analizy. Dla części autorów jest to próba pierwszej, samodzielnej pracy naukowo-badawczej, dla innych - kolejna okazja do podzielenia się przemyśleniami, ugruntowanymi wieloletnim doświadczeniem badawczym i studiami.Wszystkich połączyło i skłoniło do współpracy zainteresowanie tym samym - losem społeczeństwa obywatelskiego w zderzeniu z problemami współczesności

Effectiveness and safety of PCSK9 inhibitor therapy in patients with familial hypercholesterolemia within a therapeutic program in Poland: Preliminary multicenter data

Background: In Poland, treatment with proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors has become available free of charge in a therapeutic program. Assessed herein, is the efficacy and safety of alirocumab and evolocumab in patients with heterozygous familial hypercholesterolemia (FH).Methods: Data of 55 adult FH patients who participated in the program were analyzed upon meeting the criteria established by the Ministry of Health (low density lipoprotein cholesterol [LDL-C] above 160 mg/dL on max. tolerated statin dose and ezetimib). The efficacy of PCSK9 inhibitors in reducing LDL-C with drug administration every 2 weeks was assessed after 3 months and 1 year of therapy. A safety profile evaluation was performed at each visit. 48 patients completed the 3-month and 21 for the 1-year observation periods (34 patients treated with alirokumab and 14 with evolocumab).Results: The mean concentration of direct-measured LDL-C decreased from the initial level of 215.1 ± 74.5 mg/dL to 75.3 ± 64.1 mg/dL, i.e., by 65 ± 14% following 3 months of treatment. This effect was stable in 1-year observation (77.7 ± 72.8 mg/dL). Adverse effects were flu-like symptoms (13.0%), injection site reactions (11.1%), fatigue (5.6%) and musculoskeletal symptoms (5.6%). Seven patients failed to complete the 3-month treatment period due to side effects or non-compliance, and 1 patient failed to complete the 1-year treatment due to myalgia.Conclusions: This study confirmed high effectiveness of PCSK9 inhibitors in reducing LDL-C levels in patients with FH. Due to restrictive inclusion criteria with LDL-C threshold level > 160 mg/dL (> 4.1 mmol/L) required for participation in the therapeutic program, a relatively small number of FH patients were eligible for treatment

Inhibition of protein disulfide isomerase induces differentiation of acute myeloid leukemia cells

Acute myeloid leukemia is a malignant disease of immature myeloid cells. Despite significant therapeutic effects of differentiation-inducing agents in some acute myeloid leukemia subtypes, the disease remains incurable in a large fraction of patients. Here we show that SK053, a thioredoxin inhibitor, induces differentiation and cell death of acute myeloid leukemia cells. Considering that thioredoxin knock-down with short hairpin RNA failed to exert antiproliferative effects in one of the acute myeloid leukemia cell lines, we used a biotin affinity probe-labeling approach to identify potential molecular targets for the effects of SK053. Mass spectrometry of proteins precipitated from acute myeloid leukemia cells incubated with biotinylated SK053 used as a bait revealed protein disulfide isomerase as a potential binding partner for the compound. Biochemical, enzymatic and functional assays using fluorescence lifetime imaging confirmed that SK053 binds to and inhibits the activity of protein disulfide isomerase. Protein disulfide isomerase knockdown with short hairpin RNA was associated with inhibition of cell growth, increased CCAAT enhancer-binding protein α levels, and induction of differentiation of HL-60 cells. Molecular dynamics simulation followed by the covalent docking indicated that SK053 binds to the fourth thioredoxin-like domain of protein disulfide isomerase. Differentiation of myeloid precursor cells requires the activity of CCAAT enhancer-binding protein α, the function of which is impaired in acute myeloid leukemia cells through various mechanisms, including translational block by protein disulfide isomerase. SK053 increased the levels of CCAAT enhancer-binding protein α and upregulated mRNA levels for differentiation-associated genes. Finally, SK053 decreased the survival of blasts and increased the percentage of cells expressing the maturation-associated CD11b marker in primary cells isolated from bone marrow or peripheral blood of patients with acute myeloid leukemia. Collectively, these results provide a proof-of-concept that protein disulfide isomerase inhibition has potential as a therapeutic strategy for the treatment of acute myeloid leukemia and for the development of small-molecule inhibitors of protein disulfide isomerase

Działania opiekuńcze w profilaktyce i terapii

Praca recenzowana / Peer-reviewed paperOpieka jest potrzebna wszystkim, zarówno dzieciom, jak i dorosłym, a w sposób szczególny osobom starszym, w tym terminalnie chorym. Prezentowane w niniejszej monografi i teksty uwzględniają właśnie tę wieloczynnikową aktywność opiekuńczą. Autorami poszczególnych rozdziałów są pracownicy naukowi zaangażowani w proces edukacyjny w zakresie nauk o zdrowiu, pracujący w Krakowskiej Akademii im. Andrzeja Frycza Modrzewskiego, ale także studenci PWSZ w Tarnowie. W różnorodnym zakresie przedstawili i omówili oni główne tezy monografii

Differential regulation of serum microRNA expression by HNF1β and HNF1α transcription factors.

We aimed to identify microRNAs (miRNAs) under transcriptional control of the HNF1β transcription factor, and investigate whether its effect manifests in serum.This article is freely available via Open Access. Click on the 'Additional Link' above to access the full-text via the publisher's site.Published (Open Access