Milk thistle, Silybum marianum L. Gaertn., is a source of flavonoid taxifolin and flavonolignans - silymarin complex (silybin, silydianin, silycristin and isosilybin). Milk thistle is usually obtained by field cultivation. Alternative way for getting the active components, is the use of in vitro cultures. But the production of secondary metabolites by the in vitro cultures is low in comparison with plant. One of the possibilites how to increase this produciton is the method of elicitation. In this study, ethephon as the elicitor, in the concentrations of 500 µmol/l, 400 µmol/l, 200 µmol/l, 100 µmol/l and 50 µmol/l was used with the aim to increase secondary metabolite production in suspension and callus cultures. The effect of ethephon was compared to its inhibitor (AgNO3, 120 µmol/l). The levels of flavonolignans and taxifolin were measured by the method of HPLC. The samples were taken 24, 48, 72, 96 and 168 hours after the ethephon application and inhibitor treatment. The nutrient medium of suspension culture was also tested for the possibity of secondary metabolites releasing into medium. The highest content of flavonoid taxifolin was found in the suspension culture medium after 48 h treatment with ethephon in conc. of 400 µmol/l. The level of taxifolin was increased by 197-fold to 1,97 mg/100...

Genista tinctoria, family Fabaceae, is a potent source of isoflavonoids (genistin, genistein, daidzein, formononetin, biochanin A) with a wide spectrum of potential medical impact. Genista also contains quinolizidin alkaloids (cytisin, anagyrin, lupanin, spartein, etc.), which are toxic. The reason why in vitro cultures are used is an absence of toxic alkaloids production and higher yield of isoflavonoids in comparison with intact plant. For an increase of isoflavonoid production method of elicitation is beeing used. Isoflavonoids are studied for their phytoestrogenic effects, for which they could be used in treatment of postmenopausal symptoms and even in treatment of hormon-dependent tumours. The elicitor ethephon in concentration of 7000 µM, 700 µM and 70 µM was used in this work. The effect of ethephon inhibitor (AgNO3) in concentration of 120 µM was investigated too. Samples were examined after 24, 48, 72, 96 and 168 hours and then analysed by HPLC method. An effect of ethephon and its combination with AgNO3 was observed in callus and suspension cultures. Release of isoflavonoides into culture media was studied too. Daidzein production was the highest of all isoflavonoids in the callus culture after the treatment of ethephon in concentration of 700 µM after 96 hours (45,10 mg/g DW). The most...

The study is aimed to enhance in vitro production of secondary metabolites in Genista tinctoria L. via elicitor treatment. The different levels of elicitor concentration - selenium dioxide were utilized to affect the quantity of isoflavonoids occurred in cultures. Experiment was perfomed in callus and suspension cultures on MS nutrient media supplemented with 10 g l-1 of NAA (α-naphtylacetic acid) as growth regulator. The elicitor was added in the form of solution in concentrations of 9,012.10-3 mol l-1 ; 9,012.10-4 mol l-1 and 9,012.10-5 mol l-1 . It was exposed for 6, 12, 24, 48, 72 and 168 hours. The content of isoflavonoids was determined by HPLC in dry weight (DW) and medium. The most effective production of genistin (6,20 mg. g-1 DW, 8,30 mg. g-1 DW) in callus culture was measured. It was reached in concentrations of 9,012.10-4 mol l-1 and 9,012.10-5 mol l-1 after 168 h elicitor treatment. The second most satisfactory genistin level 5,20 mg g-1 DW was detected after elicitor application in concentration of 9,012.10-4 mol l-1 after 6 h. The content of genistein, daidzein and formononetin in callus culture was low and in the most cases equal zero compared to control samples. The content of biochanin A was equal zero compared to control samples. The most efficient daidzein production (37,10 mg...

In vitro cultures of medicinal plants - IXX The subject of this thesis is the evaluation of secondary metabolites production in in vitro cultures of Silybum marianum L. after elicitor treatment. In this study selenium dioxide as elicitor in concentrations of 9,012.10-3 mol/l; 9,012.10-4 mol/l; 9,012.10-5 mol/l was used. The samples were taken after 6, 12, 24, 48, 72 and 168 hours of elicitor treatment. The effect of elicitor was compared with control samples, which were cultured without elicitation. The content of taxifolin and flavonolignans was determined by the method of HPLC. The results showed, that almost all observed metabolites were released into a nutrient medium. Cells of callus and suspension cultures produced only small amounts of taxifolin (0.01 mg/g DW). Taxifolin and silymarin complex releasing into nutrient media was observed as in control and also in the elicitated samples of callus and suspension cultures. Selenium dioxide elicitation caused statistically significant increases in releasing taxifolin and silymarin complex into the nutrient medium. The statistically significant releasing of flavonolignans (2.2 mg/100 ml) to the medium of suspension culture was reached after 72 hours of treatment with selenium dioxide in concentration of c1 (9,012.10-3 mol/l). The statistically...

Induced pluripotent stem cells (iPSCs) have been recognised as a virtually unlimited source of stem cells that can be generated in a patient-specific manner. Due to these cells’ potential to give rise to all differentiated cell types of the human body, they have been widely used to derive differentiated cells for drug screening and disease modelling purposes. iPSCs also garner much interest as they can potentially serve as a source for cell replacement therapy. Towards the realisation of these biomedical applications, this thesis aims to address challenges that are associated with scale-up, safety and biofabrication. Firstly, the manufacture of a high number of human iPSCs (hiPSCs) will require standardised procedures for scale-up and the development of a flexible bioprocessing method, since standard adherent hiPSC culture exhibits limited scalability and is labour-intensive. While the quantity of cells that are required for cell therapy depends largely on the tissue and defect that these replacing cells are meant to correct, an estimate of 1 × 10^9 has been suggested to be sufficient for several indications, including myocardial infarction and islet replacement for diabetes. Here, the development of an integrated, microcarrier-free workflow to transition standard adherent hiPSC culture (6-well plates) to scalable stirred suspension culture in bioreactors (1 L working volume, 2.4 L maximum working volume) is presented. The two-phase bioprocess lasts 14 days and generates hiPSC aggregates measuring 198 ± 58 μm in diameter on the harvesting day, yielding close to 2 × 10^9 cells. hiPSCs can be maintained in stirred suspension for at least 7 weeks with weekly passaging, while exhibiting pluripotency-associated markers TRA-1-60, TRA-1-81, SSEA-4, OCT4, and SOX2. These cells retain their ability to differentiate into cells of all the three germ layers in vitro, exemplified by cells positive for AFP, SMA, or TUBB3. Additionally, they maintain a stable karyotype and continue to respond to specification cues, demonstrated by directed differentiation into beating cardiomyocyte-like cells. Therefore, the aim of manufacturing high hiPSC quantities was met using a state-of-the-art scalable suspension bioreactor platform. Secondly, multipotent stem cells such as induced neural stem cells (iNSCs) may represent a safer source of renewable cells compared to pluripotent stem cells. However, pre-conditioning of stem cells prior to transplantation is a delicate issue to ensure not only proper function in the host but also safety. Here, iNSCs which are normally maintained in the presence of factors such as hLIF, CHIR99021, and SB431542 were cultured in basal medium for distinct periods of time. This wash-out procedure results in lower proliferation while maintaining key neural stem cell marker PAX6, suggesting a transient pre-differentiated state. Such pre-treatment may aid transplantation studies to suppress tumourigenesis through transplanted cells, an approach that is being evaluated using a mouse model of experimental focal demyelination and autoimmune encephalomyelitis. Thirdly, biomedical applications of stem cells can benefit from recent advancements in biofabrication, where cells can be arranged in customisable topographical layouts. Employing a 3DDiscovery bioprinter, a bioink consisting of hiPSCs in gelatin-alginate was extruded into disc-shaped moulds or printed in a cross-hatch infill pattern and cross-linked with calcium ions. In both discs and printed patterns, hiPSCs recovered from these bioprints showed viability of around 70% even after 4 days of culture when loaded into gelatin-alginate solution in aggregate form. They maintained pluripotency-associated markers TRA-1-60 and SSEA-4 and continued to proliferate after re-plating. As further proof-of-principle, printed hiPSC 3D constructs were subjected to targeted neuronal differentiation, developing typical neurite outgrowth and resulting in a widespread network of cells throughout and within the topology of the printed matrix. Staining against TUBB3 confirmed neuronal identity of the differentiated cellular progeny. In conclusion, these data demonstrate that hiPSCs not only survive the 3D-printing process but were able to differentiate along the printed topology in cellular networks. / Induzierte pluripotente Stammzellen (iPSZ) stellen eine praktisch unbegrenzte Stammzellquelle dar, welche patientenspezifisch erzeugt werden kann. Da diese Zellen das Potenzial haben, alle differenzierten Zelltypen des menschlichen Körpers hervorzubringen, werden sie für die Herstellung differenzierter Zellen für Arzneimitteltests und für die Krankheitsmodellierung verwendet. Sie erfahren auch großes Interesse, weil sie als Zellquelle in der Zellersatztherapie Anwendung finden könnten. Die vorliegende Dissertation beschäftigt sich mit drei zentralen Herausforderungen, die im Rahmen der biomedizinischen Anwendung von iPSZ auftreten. Die Herstellung einer großen Zahl von humanen iPSZ (hiPSZ) erfordert die Entwicklung standardisierter Verfahren für die Skalierung, welche durch die Entwicklung einer flexiblen Bioprozessmethode realisiert werden kann. Bisher wird die Skalierbarkeit durch eine standardmäßig adhärente Zellkultur und den damit verbundenen hohen Arbeitsaufwand begrenzt. Die Menge an Zellen, die für die Zelltherapie benötigt wird, hängt stark vom Gewebetyp ab, welcher von den ersetzenden Zellen korrigiert werden soll. Berechnungen legen nahe, dass eine Anzahl 1 × 10^9 Zellen für eine Vielzahl von Indikationen ausreicht – einschließlich Myokardinfarkt und Inselzelltransplantation für Diabetes. Im Rahmen dieser Arbeit wurde ein integrierter Arbeitsablauf zur skalierbaren Zellsuspensionskultur von hiPSZ ohne Verwendung von microcarrier entwickelt, um die standardmäßig adhärente Kultur (6-Well-Platten) in Bioreaktoren (1 L Arbeitsvolumen, 2,4 L maximales Arbeitsvolumen) zu überführen. Der zweiphasige Produktionsprozess dauert 14 Tage und erzeugt hiPSZ-Aggregate mit einem finalen Durchmesser von 198 ± 58 μm, der annähernd 2 × 10^9 Zellen beinhaltet. hiPSZ können mindestens 7 Wochen lang in einer gerührten Zellsuspension bei wöchentlichem Passagieren gehalten werden, wobei sie Pluripotenz-assoziierte Marker wie TRA-1-60, TRA-1-81, SSEA-4, OCT4 und SOX2 beibehalten. Die Zellen behalten weiterhin ihre Fähigkeit, sich in vitro in Zellen mit AFP-, SMA- oder TUBB3-Immunoreaktivität und damit in Zellen aller drei Keimblätter zu differenzieren. Darüber hinaus halten sie einen stabilen Karyotyp aufrecht und reagieren auf gezielt eingesetzte externe Differenzierungsstimuli, wie durch eine gezielte Differenzierung in schlagende Kardiomyozyten-ähnliche Zellen demonstriert werden konnte. Somit wurde das Ziel, eine großen Anzahl hiPSCs herzustellen, mit einer hochmodernen, skalierbaren Suspensionsbioreaktorplattform erreicht. Multipotente Stammzellen wie induzierte neurale Stammzellen (iNSZ) gelten verglichen mit iPSZ als sicherere Zellquelle für Ersatztherapien. Die Vorkonditionierung von Stammzellen vor der Transplantation ist jedoch ein heikles Thema, da sowohl die einwandfreie Funktion im Wirtsgewebe als auch Sicherheit gewährleistet werden müssen. Im Rahmen dieser Arbeit wurden iNSZ, die normalerweise im Kulturmedium mit Faktoren wie hLIF, CHIR99021 und SB431542 gehalten werden, für eine definierte Zeitspanne in basalem Medium kultiviert. Die Vorbehandlung führt zu einer geringeren Proliferation, jedoch unter Erhalt der Expression des wichtigen neuralen Stammzellmarkers PAX6, was auf einen transienten vordifferenzierten Zustand hindeutet. Eine solche Vorbehandlung könnte bei zukünftigen Transplantationsstudien angewandt werden, um die Tumorentstehung durch transplantierte Zellen zu unterdrücken. Dieser Ansatz wird in Zukunft mit einem Mausmodell der experimentellen fokalen Demyelinisierung und der autoimmunen Enzephalomyelitis untersucht. Schließlich kann die Zellersatztherapie von den jüngsten Fortschritten in der Biofabrikation profitieren, bei der die Zellen durch das Drucken in anpassbare topographische Profile angeordnet werden können. Mit einem 3DDiscovery Biodrucker wurde eine Biotinte bestehend aus Gelatine-Alginat und hiPSZ in scheibenförmig extrudiert oder in einem Kreuzschraffurmuster gedruckt und mittels Kalziumionen-Zugabe vernetzt. Gedruckte hiPSZ zeigten auch nach 4 Tagen Kultivierung eine Lebensfähigkeit von etwa 70 % und weiterhin das Auftreten der Pluripotenz-assoziierten Marker TRA-1-60 und SSEA-4. Zudem konnten sie sich anschließend mit standardmäßig adhärenter Zellkultur weiter vermehren. Zudem konnte gezeigt werden, dass die gedruckten Konstrukte einer gezielten neuronalen Differenzierung unterzogen werden können, die zu einem typischen Neuritenauswuchs und zu einer weitreichenden interzellulären Vernetzung durch und innerhalb der Topologie der gedruckten Matrix führte. Die Färbung gegen TUBB3 bestätigte die neuronale Identität der differenzierten Zellen. Zusammenfassend zeigen diese Daten, dass bei Verwendung des in dieser Studie erarbeiteten Protokolls hiPSZ nicht nur den 3D-Druckprozess überleben, sondern auch entlang der gedruckten 3D Topologie in Netzwerke Neurone differenzieren können.

Cellulose is a biopolymer of great relevance in the plant cell walls, where it constitutes the most important skeletal component. Cellulose is also an important raw material in the pulp- and paper, forest, and textile industries, among others. Cellulose biosynthesis in particular, and xylogenesis in general are processes which are currently poorly understood. Yet, research in cellulose synthesis is progressing and different applications of cellulose, mainly cellulose derivatives for e.g. pharmaceuticals and coatings, are constantly emerging. This thesis depicts how cellulose synthase (CesA) genes in Populus were identified and characterized by gene expression- and bioinformatics analyses. Within an EST database of more than 100,000 clones from wood forming tissues of three different Populus taxa, ten CesA genes were identified in Populus tremula x tremuloides. Subsequent gene expression analyses by using microarrays and real-time PCR experiments in woody tissues, revealed distinct regulation patterns among the genes of interest. This enabled proper classification and characterization of the secondary cell wall related CesA genes, in particular. Bioinformatic analyses of the genome sequence of Populus trichocarpa further provided a complete picture of the number of putative CesA genes retained after several duplication events during tree evolution. In contrast to the previously reported set of ten 'true' CesA genes in many other plant species, the genome of P. trichocarpa encodes 18 putative proteins, which could be assembled into nine groups according to their sequence similarities. Interestingly, studies in the EST database suggested that paralogs within at least two groups have corresponding orthologs in P. tremula x tremuloides, which are furthermore transcribed. This implies that at least some of the duplicated genes have remained functional, or may have acquired a modified function. By focusing on the CesA genes associated with secondary cell wall formation, cellulose synthesis was also studied in poplar cell suspension cultures. Selection of CesA enriched material was performed by determining expression intensities of the CesA genes using RT-PCR, whereupon membrane protein extraction was initiated. CesA proteins are part of large cellulose synthesizing complexes in the plasma membrane. Subsequent proteomic approaches comprised partial purification of these cellulose synthesizing complexes from protein enriched culture material and in vitro cellulose synthesis experiments. De novo synthesized material was successfully characterized and the acquired yields were as high as 50% cellulose (compared to previously reported yields of 30% in other plant systems) of the total in vitro synthesized product. Elevated CesA gene expression levels can thus be correlated to increased protein activity in poplar cell suspension cultures. In addition, antibodies raised against CesA antigens were used in Western blot analyses comprising samples along the protein extraction- and purification procedure. Proteins with corresponding molecular weight to the theoretical 120kDa of CesA proteins were recognized by a range of different specific antibodies. The study demonstrates that poplar cell suspension cultures can provide a valuable model system for studies of cellulose synthesis and different aspects of xylogenesis. / QC 20101005

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Candidate: Tereza Ošťádalová Supervisor: Doc. PharmDr. Lenka Tůmová, CSc. Title of diploma thesis: The selenium effect on secondary metabolites production in in vitro cultures of medicinal plants - II. Key words: callus, suspension culture, abiotic elicitor, selenium, Fagopyrum In vitro plant cultures usually produce only a small amount of secondary metabolites. The method of elicitation is one of the options how to increase the production of these substances. The effect of selenium as abiotic elicitor on rutin production in callus and suspension culture of Fagopyrum esculentum Moench., variety Pyra was observed in this study. The release of rutin into the nutrient medium was studied as well. The cultivation was performed in Murashige and Skoog (MS) nutrient medium which was enriched with 1 mg/l of 2,4-dichlorophenoxyacetic acid. The solution of selenium in three different concentrations (c1 = 9.012.10-3 mol/l, c2 = 9.012.10-4 mol/l, c3 = 9.012.10-5 mol/l) was used. The samples were taken after 6, 12, 24, 48, 72 and 168 hours of elicitor treatment. The rutin content was determined by high-performance liquid chromatography (HPLC). The elicitation led to increasing of rutin amount in callus and also in suspension...

Higher plants represent an important source of valuable substances, so called secondary metabolites, which can be obtained through explant cultures of plants. Elicitation is a method of increasing the secondary metabolites production. This study aims to evaluate the secondary metabolites production in Fagopyrum esculentum variety Spacinska cultures in vitro after abiotic elicitor treatment. The experiment was focused on alteration of rutin production in callus and suspension cultures of F. esculentum var. Spacinska after selenium application. Murashige and Skoog nutrient medium supplemented with 1 mg l-1 2,4-dichlorophenoxyacetic acid was used for the cultivation. Selenium solutions of various concentrations (c1 = 9.012×10-3 mol l-1 , c2 = 9.012×10-4 mol l-1 , c3 = 9.012×10-5 mol l-1 ) were affecting the cultures for 6, 12, 24, 48, 72 and 168 hours. The content of rutin was determined by HPLC. The release of secondary metabolites into the nutrient medium was studied as well. After elicitor application, the rutin production increased in both callus and suspension cultures. Higher levels of rutin content were detected in callus culture. The maximum rutin content (0.6 mg g-1 DW) was reached after 12 h of elicitor treatment of c2 concentration in callus culture. Concerning suspension culture, the...

Tissue engineering is a dichotomy of scaffold-based and scaffold-free cultures. Scaffold-based cultures form highly organotypic structures but with low uniformity and throughput. Conversely, scaffold-free cultures create consistently sized and shaped cell aggregates with limited spheroid-like structure and function, thus restricting their use for accurate disease modeling. We hypothesized that combining aspects of each culturing format, we would produce highly organotypic structures of consistent size and shape for use in pulmonary modeling. First, to improve on culture consistency and output, we created a novel easily scalable, minimalistic design for a micropatterned hydrogel dish that increases reliability and efficiency in 3D cell culture. This dish design features three times more efficient media change relative to commercially available plates. Moreover, we discovered that formation of consistently sized and shaped cell aggregates depended on hydrogel stiffness. Second, we developed a biocompatible 3D printing resin using poly(ethylene glycol) diacrylate (PEGDA) monomer with avobenzone as the UV absorber instead of 2-nitrophenyl phenyl sulfide (NPS). The polymerized resin could be surface activated to promote cell adhesion. This resin could be used in high-resolution printing of miniature devices for microfluidic and nanofluidic cell culture and cell assays. Third, we show a unique improvement on current methods to produce organotypic aggregates via suspension culture. By using soluble non-gelling concentration of basement membrane (BM), we created an organotypic lung model from three stable cells representing epithelial, vascular, and fibroblast cell populations within 14 days of culture. We observed that soluble BM promotes emergence of lumina comparable to mammalian lung airspaces. Using hypoxia induction techniques, we provide evidence for formation of branching, perfusable vasculature in pulmonary aggregates supplemented with soluble BM. Aside from these structural traits, we observed increased proliferation, survival, and 3D growth of aggregates. These results were supported by proteomic studies. As proof of concept, we applied this method in modeling of lung fibrosis using bleomycin induction followed by testing one investigational antifibrotic drug. Our results demonstrate a novel 3D culture method that creates organotypic models from stable cell lines. We anticipate this technology to pioneer creation of novel suspension-based organoids fostering consistent, expedited 3D culture. In summary, these three technologies highlighted in this dissertation improved on the 3D culture status quo. We view these technologies to have the potential to expedite creation of patient-derived organoids for personalized drug screening using lung-on-a-chip assays.

Plant cell and tissue cultivations are of growing interest for the production of structurally complex and expensive plant-derived products, especially in pharmaceutical production. Problems with up-scaling, low yields and high-priced process conditions result in an increased demand for models to provide comprehension, simulation, and optimization of production processes. In the last 25 years, many models have evolved in plant biotechnology; the majority of them are specialized models for a few selected products or nutritional conditions. In this article we review, delineate, and discuss the concepts and characteristics of the most commonly used models. Therefore, the authors focus on models for plant suspension and submerged hairy root cultures. The article includes a short overview of modeling and mathematics and integrated parameters, as well as the application scope for each model. The review is meant to help researchers better understand and utilize the numerous models published for plant cultures, and to select the most suitable model for their purposes.