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Over-expression of the vesicular stomatitis virus glycoprotein (VSVG) in mammalian cells can induce the formation of VSVG-pseudotyped vesicles (named “gesicles”) from membrane budding. Its use as a nucleic acid delivery tool is still poorly documented. Naked-plasmid DNA can be delivered in animal cells with gesicles in presence of hexadimethrine bromide (polybrene). However, little is known about gesicle manufacturing process and conditions to obtain successful nucleic acid delivery. In this study, gesicles production process using polyethylenimine (PEI)- transfected HEK293 cells was developed by defining the VSVG-plasmid concentration, the DNA:PEI mass ratio, and the time of gesicle harvest. Furthermore, parameters described in the literature relevant for nucleic acid delivery such as (i) component concentrations in assembly mixture, (ii) component addition order, (iii) incubation time, and (iv) polybrene concentration were tested by assessing the transfection capacity of the gesicles complexed with a green fluorescent protein (GFP)-coding plasmid. Interestingly, freezing/thawing cycles and storage at + 4 °C, − 20 °C, and − 80 °C did not reduce gesicles’ ability to transfer plasmid DNA. Transfection efficiency of 55% and 22% was obtained for HeLa cells and for hard-to-transfect cells such as human myoblasts, respectively. For the first time, gesicles were used for delivery of a large plasmid (18-kb) with 42% of efficiency and for enhanced green fluorescent protein (eGFP) gene silencing with siRNA (up to 60%). In conclusion, gesicles represent attractive bioreagents with great potential to deliver nucleic acids in mammalian cells.

Flaxseed meal, a byproduct of flaxseed oil extraction, was treated as low-value agrowaste for a long time despite its high protein content. Flaxseed meal has recently garnered increasing interest as a source of proteins and other bioactive compounds with positive impacts on human health. The aim of this study was to investigate the in vitro biological potential of flaxseed protein hydrolysates (FPH). Three FPHs were prepared using three hydrolytic enzymes: Alcalase, Neutrase and Protamex. The molecular weight profile of peptides contained in the hydrolysates was determined by size exclusion chromatography (SEC). The oxygen radical absorbance capacity (ORAC) assay was used to determine the peptide antioxidant capacity, while proliferative effects were studied in two cell lines: HeLa and HaCaT. The latter was also used to determine the protective effect of the FPH during induced oxidative stress. Alcalase showed the highest proteolytic activity, while the produced flaxseed protein hydrolysate (FPH-A) exhibited the strongest antioxidant potential. FPH-A had cytotoxic effects at 10 mg/mL in HeLa cells, but it stimulated HaCaT cell growth. Moreover, a mild protective effect of FPH-A was detected in HaCaT cells after induction of oxidative stress. Flaxseed protein hydrolysates obtained by Neutrase (FPH-N) and Protamex (FPH-P) have less pronounced or no potential at all, with respect to their antioxidative or antiproliferative activity. Therefore, to increase value-added utilization of flaxseed meal we suggest further research on hydrolysate obtained by Alcalase.

U procesima s kulturama životinjskih stanica koristi se medij za uzgoj koji sadrži osnovne nutrijente poput glukoze, amino-kiselina, soli te hormone i faktore rasta iz seruma životinjskog porijekla. Zbog nedostataka primjene seruma u procesima s kulturama stanica (npr. rizik prijenosa virusa, varijacije u sastavu, otežana izolacija proizvoda) pristupilo se razvoju medija bez seruma u koje se često dodaju i hidrolizati proteina biljnog porijekla. Hidrolizati sadrže aminokiseline, oligopeptide, lipide, vitamine i ostale spojeve koji potiču proliferaciju i produktivnost stanica u kulturi. U ovom radu dan je kratak pregled priprave i najvažnijih bioloških svojstava proteinskih hidrolizata s naglaskom na primjenu biljnih hidrolizata kao sastojaka medija za uzgoj životinjskih stanica u proizvodnji biofarmaceutika., Culture media containing essential nutrients like glucose, amino-acids and salts, as well as hormones, growth factors added by animal blood serum is required for bioprocesses with animal cells. Due to the disadvantages of serum use (e.g. risk of virus transmission, variation in composition, complex product isolation), the development of serum-free media supplemented with plant protein hydrolysates took place about two decades ago. The hydrolysates contain amino acids, oligopeptides, lipids, vitamins and other compounds that promote cell culture proliferation and productivity. This paper gives a brief overview of the preparation and the most important biological properties of protein hydrolysates with emphasis on the use of plant hydrolysates as components of animal cell culture media in the production of biopharmaceuticals.

Povećanje broja stanovnika vodi k povećanoj potražnji za hranom. Zato je vrlo važno baviti se usklađivanjem povećane proizvodnje hrane sa smanjenjem štetnog utjecaja na okoliš kojeg ima globalno širenje poljoprivrede. Novo područje biotehnologije, nazvano stanična poljoprivreda, moglo bi doprinijeti rješavanju ovog problema. Stanična poljoprivreda nudi drugačije proizvodne sustave za mnoge proizvode životinjskog i biljnog podrijetla koji se danas dobivaju stočarstvom ili poljodjelstvom. Meso i mlijeko dva su takva proizvoda, a proizvodnja njihovih analoga primjenom stanične poljoprivrede cilj je sve većeg broja tvrtki širom svijeta. One razvijaju tehnologiju oslanjajući se na iskustva stečena u tkivnom inženjerstvu i bioprocesima sa životinjskim stanicama. U ovom radu donosimo pregled novih tehnologija koje se temelje na uzgoju kulture stanica za dobivanje tzv. in vitro kultiviranog mesa i analoga mlijeka. Također raspravljamo o kvaliteti navedenih proizvoda te potrebama i održivosti njihove proizvodnje., Increasing population means increased global food demand. Therefore we are facing a great challenge in harmonizing the production of food with the need to reduce adverse environmental impacts of expanding agriculture. The problem could be solved by adopting a new field in biotechnology known as cellular agriculture. This agriculture can provide alternative manufacturing systems for many animal and plant-based products. The products mimicking meat and milk, are a major target of a growing number of start-up companies worldwide. These companies develop their technology relying on know-how from tissue engineering and animal cell bioprocessing. Here we present an overview of the cell culture-based technologies for in vitro meat and milk analogues production. We also discuss the quality of the products as well as the production requirements and sustainability., Eine wachsende Bevölkerung bedeutet eine steigende weltweite Nachfrage nach Nahrungsmitteln. Daher stehen wir vor der großen Herausforderung, die Produktion von Nahrungsmitteln mit der Notwendigkeit in Einklang zu bringen, die negativen Auswirkungen der expandierenden Landwirtschaft auf die Umwelt zu reduzieren. Das Problem könnte durch die Einführung eines neuen Zweigs der Biotechnologie, der sogenannten zellulären Landwirtschaft, gelöst werden. Diese Landwirtschaft bietet alternative Herstellungssysteme für viele tierische und pflanzliche Produkte, die heutzutage in der Viehzucht und Landwirtschaft hergestellt werden. Fleisch und Milch sind zwei solcher Produkte; die Herstellung von deren Ersatzprodukten anhand der zellulären Landwirtschaft ist ein wichtiges Ziel zahlreicher Unternehmen weltweit. Diese Unternehmen entwickeln ihre Technologie auf der Grundlage des Know-hows aus dem Tissue Engineering und der Bioprozessierung von tierischen Zellen. Hier geben wir einen Überblick über die zellkulturbasierten Technologien für die In-vitro-Produktion von Fleischund Milchanaloga. Wir erörtern auch die Qualität der Produkte und die Nachhaltigkeit der Produktion., El aumento de la población conduce a una mayor demanda de alimentos. Por eso es muy importante abordar la armonización del aumento de la producción de alimentos con la reducción del impacto nocivo sobre el medio ambiente que tiene la expansión global de la agricultura. Una nueva área de la biotecnología, llamada agricultura celular, podría ayudar a abordar este problema. La agricultura celular ofrece diferentes sistemas de producción para muchos productos de origen animal y vegetal que se obtienen hoy en día por la ganadería o la agricultura. La carne y la leche son dos de esos productos, y la producción de sus sustitutos mediante agricultura celular es el objetivo de un número creciente de empresas en todo el mundo. Desarrollan tecnología basada en la experiencia adquirida en ingeniería de tejidos y bioprocesos de células animales. En este artículo presentamos una descripción general de las nuevas tecnologías basadas en la cultivación de los cultivos celulares para obtener in vitro la llamada carne cultivada y los análogos de leche. También discutimos la calidad de estos productos y la sostenibilidad de su producción., L’aumento della popolazione mondiale porta inevitabilmente ad un aumento della domanda alimentare. Ecco perché è molto importante accompagnare l’aumento della produzione alimentare mondiale con la riduzione dell’impatto nocivo sull’ambiente che l’espansione globale dell’agricoltura comporta. Una nuova area della biotecnologia, chiamata agricoltura cellulare, potrebbe aiutare a risolvere questo problema. L’agricoltura cellulare offre diversi sistemi di produzione per molti prodotti di origine animale e vegetale che oggi si ottengono dall’allevamento o dall’agricoltura tradizionali. La carne e il latte sono due di questi prodotti, e la produzione dei loro analoghi mediante l’agricoltura cellulare è l’obiettivo di un numero crescente di aziende in tutto il mondo. I loro sforzi si concentrano intorno allo sviluppo di una tecnologia basata sull’esperienza acquisita nell’ingegneria tissutale e nei processi cellulari animali. In questo articolo presentiamo una panoramica delle nuove tecnologie basate sull’agricoltura cellulare funzionali alla produzione degli analoghi della carne e del latte coltivati in vitro. Discutiamo anche della qualità di questi prodotti e della sostenibilità della loro produzione.

In this paper, the effect of different collector geometry on the PCL scaffold architecture and cell viability was investigated. PCL scaffolds with antibiotic CefuroximVR , 15%wt. and 20%wt., were electrospun and characterized by ATR-FTIR, while thermal stability were observed by DSC and TG. PCL/15%CFUVR scaffolds were electrospun on six 3D printed collectors, three ribbed and three mesh geometries, to produce the most favourable structure for cells adhesion and proliferation. SEM study and MTT assay revealed that collectors with the widest slot in both ribbed and mesh geometries exhibited the most favourable structure to enhance cells adhesion. In general, cells spreading on the surface and cells viability proved to be better for the ribbed collectors. The study showed that by controlling the collector geometry and the scaffold three-dimensional structure, it is possible to control cell attachment and differentiation and, consequently, to adjust them for the special tissue demands in the regeneration process.

Polilaktidna kiselina (PLA) i bakterijska nanoceluloza (BNC) zbog svoje biorazgradljivosti, biokompatibilnosti i netoksičnosti imaju velik potencijal za primjenu u biomedicini. Cilj ovog rada bio je pripraviti i ispitati biokompozit PLA/BNC. Istražen je utjecaj BNC-a na morfološku strukturu, kemijski sastav, toplinska svojstva, toplinsku postojanost i hidrofobnost PLA te zasijavanje i rast stanica biokompozita PLA/BNC primjenom pretražnog elektronskog mikroskopa (SEM), infracrvene spektroskopije (FTIR), diferencijalne pretražne kalorimetrije (DSC) i termogravimetrijske analize (TGA) te određivanjem kontaktnog kuta i metodom MTT. Dodatkom BNC-a u PLA dolazi do pomaka staklišta (Tg) prema nižim temperaturama, što ukazuje na veću pokretljivost amorfne faze PLA te porasta stupnja kristalnosti zbog nukleacijskog učinka celuloze. Početak toplinske razgradnje pomaknut je na niže temperature u odnosu na čisti PLA, što ukazuje na smanjenje toplinske postojanosti PLA dodatkom BNC-a. Biokompozit PLA/BNC pokazuje poroznu, vlaknastu strukturu. Test zasijavanja stanica pokazao je da je biokompozit PLA/BNC pogodan za prihvaćanje i rast humanih stanica, pa je prema tome potencijalno primjenjiv u regenerativnoj medicini i tkivnom inženjerstvu.

Natural polymer-based scaffolds are generally considered as favourable matrices for the adhesion and growth of cells in tissue repair. One of the most popular materials in this respect is silk fibroin, known for its wide usage in biomedical applications. This work focuses on the development of electrospun scaffolds based on poly(ε-caprolactone) (PCL) and silk fibroin (SF) evaluated regarding the SF effect on their morphology, surface wetting ability, thermal properties, and HaCaT model cell line biocompatibility. The study revealed that the lowest PCL/SF concentration resulted in highest bead-like morphology formation, relatively thick fibers with the presence of random beads in the case of PCL, while uniform and thinner fibers in the case of increasing PCL/SF content scaffolds. The addition of SF reduced the degree of crystallinity in the PCL due to the less organized crystal structure, and decreased its thermal stability. Both SEM and MTT analyses showed cell presence on all scaffolds three days after cell seeding. Although SF improved PCL hydrophilicity, as shown quantitatively by the MTT assay for improved cytocompatibility properties, more structured electrospun PCL/SF scaffold strategies are required.

Electrospun materials are good candidates for the design of tissue regeneration scaffolds as they can simulate the natural surroundings of tissue cells. The study proposes electrospun polycaprolactone (PCL)/cefuroxime (CFU) scaffolds for human cell culture and investigates the influence of the antibiotic content on scaffold morphology, thermal and mechanical properties. The increase in the CFU concentration resulted in the reduction of fiber diameter and number of deformations. It also influenced the reduction of scaffold thermal enthalpies and improved scaffold break strength. With regard to cell growth, the scaffolds showed precedence in greater colonization of the HeLa cells. Finally, these scaffolds showed compatibility with standard human cell lines, and thus they can be used for the repair of damaged tissues.

Limbal Stem Cell Deficiency (LSCD) is a very serious and painful disease that often results in impaired vision. Cultivation of limbal stem cells for clinical application is usually performed on carriers such as amniotic membrane or surgical fibrin gel. Transplantation of these grafts is associated with the risk of local postoperative infection that can destroy the graft and devoid therapeutic benefit. For this reason, electrospun scaffolds are good alternatives, as proven to mimic the natural cells surroundings, while their fabrication technique is versatile with regard to polymer functionalization and scaffolds architecture. This study considers the development of poly(&epsilon, caprolactone) (PCL) immune-compatible and biodegradable electrospun scaffolds, comprising cefuroxime (CF) or titanium dioxide (TiO2) active components, that provide both bactericidal activity against eye infections and support of limbal stem cells growth in vitro. The PCL/CF scaffolds were prepared by blend electrospinning, while functionalization with the TiO2 particles was performed by ultrasonic post-processing treatment. The fabricated scaffolds were evaluated in regard to their physical structure, wetting ability, static and dynamic mechanical behaviour, antimicrobial efficiency and drug release, through scanning electron microscopy, water contact angle measurement, tensile testing and dynamic mechanical analysis, antimicrobial tests and UV-Vis spectroscopy, respectively. Human limbal stem cells, isolated from surgical remains of human cadaveric cornea, were cultured on the PCL/CF and PCL/TiO2 scaffolds and further identified through immunocytochemistry in terms of cell type thus were stained against p63 marker for limbal stem cells, a nuclear transcription factor and cytokeratin 3 (CK3), a corneal epithelial differentiation marker. The electrospun PCL/CF and PCL/TiO2 successfully supported the adhesion, proliferation and differentiation of the cultivated limbal cells and provided the antimicrobial effect against Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans.

Subcritical water extraction (SWE) has become a popular green extraction technique for the isolation of different classes of compounds from natural matrices. Low price, safety and green character of water, good yields of target compounds and reduced energy consumption, make this technique favorable for potential industrial applications. The purpose of this study was to evaluate antioxidant, antimicrobial and cytotoxic activity of four medicinal plants traditionally used in folk medicine of Serbia. Black mulberry (Morus nigra L.), wall germander (Teucrium chamaedrys L.), wild geranium (Geranium macrorrhizum L.) and comfrey (Symphytum officinale L.) were extracted by subcritical water at different temperatures. Antioxidant activity of the extracts was defined by conventional spectrophotometric methods, such as the total phenolic content (TPC), DPPH-radical scavenging activity (DPPH-RSA), ferric reducing antioxidant power (FRAP) and total antioxidant capacity (TAC) assessed by a DNA-based sensor. Additionally, the main phenolic compounds contributing to the antioxidant activity of the produced extracts were also identified and quantified by high performance liquid chromatography with diode array detection (HPLC-DAD). Antimicrobial properties of extracts were evaluated against eight microbial strains. Furthermore, the cytotoxic activity was observed for two human cancer cell lines and a cell line derived from murine fibroblast., The project 6818–Transnational Cooperation, Agreement between Portugal (FCT) and Serbia (MSTD) is acknowledged. This work was also funded by project TR 31014 financially supported by the Serbian Ministry of Education, Science and Technological Development. Financial support from project within the bilateral cooperation between the Republic of Serbia and the Republic of Croatia is gratefully acknowledged. Authors are also grateful for financial support from project NORTE-01-0145-FEDER-000011–311 Qualidade e Segurança Alimentar—uma abordagem (nano) tecnológica. The financial support from FCT/MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT2020 through the project UID/QUI/50006/2013–POCI/01/0145/FEDER/007265, is also acknowledged. M.F. Barroso (SFRH/BPD/78845/2011) and Manuela M. Moreira (SFRH/BPD/97049/2013) are grateful to Fundação para a Ciência e Tecnologia for their postdoctoral fellowships.

Gripa (ili influenca) je virusna, infektivna i respiratorna bolest koja godišnje zarazi do milijardu ljudi diljem svijeta. Pojavljuje se svake godine kao sezonska gripa, no u povijesti se svakih nekoliko desetaka godina javljala i kao pandemija. Njezin nepovoljan učinak na pojedinca i društvo može se spriječiti upotrebom cjepiva, tj. cijepljenjem. Prvo odobreno cjepivo protiv gripe proizvedeno je u embrioniranom kokošjem jajetu prije skoro 80 godina, a po sličnom tehnološkom načelu proizvode se i današnja cjepiva. Napredak u razumijevanju stanične biologije te unaprjeđenje proizvodnih postupaka pomoću kulture stanica omogućilo je stručnjacima prijelaz s konvencionalnih na nove, produktivnije i brže proizvodne postupke. U radu se najprije razmatra epidemiloški aspekt gripe, struktura virusa gripe, a zatim dosezi i izazovi u suvremenoj proizvodnji cjepiva protiv gripe. U završnom dijelu ističu se potrebe i trendovi u izradi univerzalnog cjepiva protiv gripe., Influenza (flu) is a viral, contagious, respiratory disease that annually infects up to a billion people worldwide. It appears each year as a seasonal flu, but through history, it appeared as pandemic illness once in every several decades. Its harmful effects on human health and consequently to the society can be prevented by vaccination. The first approved influenza vaccine was made almost 80 years ago using embryonated hen eggs and the same technology is still in use for most available vaccines today. The development of cell biology and improvement of cell culture manufacturing techniques enabled the transition from conventional to novel, more efficient vaccine production methods. In this work we describe the epidemiology of influenza, influenza virus structure as well as current influenza vaccine manufacturing approaches. A brief overview of universal vaccine development and its urgency is discussed in the final section of the paper.

Polilaktidna kiselina (PLA) i bakterijska nanoceluloza (BNC) zbog svoje biorazgradljivosti, biokompatibilnosti i netoksičnosti imaju velik potencijal za primjenu u biomedicini. Cilj ovog rada bio je pripraviti i ispitati biokompozit PLA/BNC. Istražen je utjecaj BNC-a na morfološku strukturu, kemijski sastav, toplinska svojstva, toplinsku postojanost i hidrofobnost PLA te zasijavanje i rast stanica biokompozita PLA/BNC primjenom pretražnog elektronskog mikroskopa (SEM), infracrvene spektroskopije (FTIR), diferencijalne pretražne kalorimetrije (DSC) i termogravimetrijske analize (TGA) te određivanjem kontaktnog kuta i metodom MTT. Dodatkom BNC-a u PLA dolazi do pomaka staklišta (Tg) prema nižim temperaturama, što ukazuje na veću pokretljivost amorfne faze PLA te porasta stupnja kristalnosti zbog nukleacijskog učinka celuloze. Početak toplinske razgradnje pomaknut je na niže temperature u odnosu na čisti PLA, što ukazuje na smanjenje toplinske postojanosti PLA dodatkom BNC-a. Biokompozit PLA/BNC pokazuje poroznu, vlaknastu strukturu. Test zasijavanja stanica pokazao je da je biokompozit PLA/BNC pogodan za prihvaćanje i rast humanih stanica, pa je prema tome potencijalno primjenjiv u regenerativnoj medicini i tkivnom inženjerstvu. Ovo djelo je dano na korištenje pod licencom Creative Commons Imenovanje 4.0 međunarodna., Polylactic acid (PLA) and bacterial nanocellulose (BNC) are promising materials in medicine due to their biodegradability, biocompatibility, and non-toxicity. The aim of this work was to prepare and characterize the PLA/BNC biocomposite. Morphology, chemical composition, thermal properties, thermal stability, hydrophobicity and cell seeding, and growth of the PLA/BNC biocomposite were characterized by means of scanning electron microscopy (SEM), Fourier transform infrared spectra (FTIR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), contact angle, and MTT method. DSC showed that the glass transition temperature (Tg) of PLA decreased with the addition of BNC due to higher mobility of amorphous PLA phase. The degree of crystallinity increased due to nucleation effect of cellulose. With the addition of BNC, the thermal stability of biocomposite decreased. The PLA/BNC biocomposite exhibited a porous, fibrous structure. The cell seeding test showed the PLA/BNC biocomposite to be suitable for growth of human cells, and therefore, potentially applicable in regenerative medicine and tissue engineering. This work is licensed under a Creative Commons Attribution 4.0 International License.

Expression of recombinant proteins exerts stress on cell culture systems, affecting the expression of endogenous proteins, and contributing to the depletion of nutrients and accumulation of waste metabolites. In this work, 2D-DIGE proteomics was employed to analyze differential expression of proteins following stable transfection of a Chinese Hamster Ovary (CHO) cell line to constitutively express a heavy-chain monoclonal antibody. Thirty-four proteins of significant differential expression were identified and cross-referenced with cellular functions and metabolic pathways to identify points of cell stress. Subsequently, 1D-(1)H NMR metabolomics experiments analyzed cultures to observe nutrient depletion and waste metabolite accumulations to further examine these cell stresses and pathways. From among fifty metabolites tracked in time-course, eight were observed to be completely depleted from the production media, including: glucose, glutamine, proline, serine, cystine, asparagine, choline, and hypoxanthine, while twenty-three excreted metabolites were also observed to accumulate. The differentially expressed proteins, as well as the nutrient depletion and accumulation of these metabolites corresponded with upregulated pathways and cell systems related to anaplerotic TCA-replenishment, NADH/NADPH replenishment, tetrahydrofolate cycle C1 cofactor conversions, limitations to lipid synthesis, and redox modulation. A nutrient cocktail was assembled to improve the growth medium and alleviate these cell stresses to achieve a ∼75% improvement to peak cell densities.

Cells grown on bioactive matrices have immensely advanced many aspects of biomedical research related to drug delivery and tissue engineering. Our main objective was to perform simple evaluation of the structural and biotic qualities of cell scaffolds made of affordable biomaterials for liver cell line (HepG2) cultivation in vitro. In this work the electrospun matrix made of synthetic polyester poly(&epsilon, caprolactone) (PCL) was compared with the natural protein-based extracellular matrix isolated from porcine liver (ECM). Mechanical and structural analysis showed that ECM was about 12 times less resistant to tensile stress while it had significantly larger pore size and twice smaller water contact angle than PCL. Bioactivity assessment included comparison of cell growth and transfection efficiency on cell-seeded scaffolds. Despite the differences in composition and structure between the two respective matrices, the rate of cell spreading and the percentage of transfected cells on both scaffolds were fairly comparable. These results suggest that in an attempt to produce simple, cell carrying structures that adequately simulate the natural scaffold, one can rely on PCL electrospun mats.

Nastojanje suvremene prehrambene industrije je što učinkovitije iskoristiti organske ostatke nastale preradom sirovina u prodajne proizvode. Primjer takvog organskog ostatka je i uljna pogača, koja zaostaje nakon ekstrakcije ulja iz sjemenki lana, a zbog svog sastava proteina predstavlja potencijalnu sirovinu za primjenu u tehnologiji uzgoja stanica. Stoga je iz uljne pogače lana izoliran proteinski izolat te je ispitan njegov utjecaj na rast CHO-E i HEK-293T staničnih linija, kao i na produktivnost CHO-E stanične linije. Dokazano je da dodatak proteinskog izolata u hranjivi medij u vrlo niskim koncentracijama (0,1 g L-1 i/ili 0,2 g L-1) ima pozitivan učinak na rast obje stanične linije, kao i na produktivnost CHO-E stanične linije. Međutim, dodatak većih koncentracija izolata od navedenih ne utječe pozitivno na rast obje stanične linije, odnosno na njih djeluje inhibitorno.

Food proteins from different sources can provide beneficial effects on human health by releasing the bioactive peptides that are integral part of their native structure. In this study, we tested the biological potential of hempseed protein hydrolysates (HPHs) obtained from hempseed cake protein isolate. The HPHs were prepared by enzyme hydrolysis using three different proteases of microbial origin: Alcalase®, Neutrase® and Protamex®. The antioxidant activity of the obtained hydrolysates was determined by oxygen radical absorbance capacity (ORAC) assay, while the proliferative effects on normal (HaCaT) and cancer (HeLa) cells were determined by the CellTiter 96® AQueous One Solution Reagent (MTS) assay. HPHs showed dose-dependent antiproliferative effects on HeLa cells and stimulatory effects on the proliferation of HaCaT cells. HPH obtained by Neutrase® (HPH-N) showed the highest antioxidant activity expressed as an ORAC value. The protective effect of HPH-N on H2O2-induced oxidative stress in normal and cancer cells was evaluated and 1 mg/mL of HPH-N significantly reduced the formation of intracellular reactive oxygen species (ROS) in both cell lines. The obtained results indicate the benefits of HPHs as potential natural antioxidants for the food industry and contribute to the growing trend of utilizing hempseed by-products.

The study focuses on the fabrication and properties evaluation of dual functional electrospun polycaprolactone scaffolds incorporating antibiotic drug cefuroxime and having custom tailored topography for the support of human cells to be used in tissue regeneration. The scaffolds were evaluated in regard to their morphology, total porosity, thermal properties, tensile properties and cells adhesion/growth, thus to define the effect of both drug content and physical architecture on the scaffolds properties. Both the addition of the drug and the scaffolds custom topography resulted in higher total porosity (up to 90 %), while the fibers diameter highest reduction was up to 73 %. The type of 3D printed collector influenced scaffolds tensile properties, thus the increase of the tensile strength was due to the change of the central cylinder height in the hexagonal geometry of the collectors. Cells distribution and spreading on the electrospun scaffolds after 72 hours, showed that scaffolds with custom topography have more evenly scattered cells than the ones with random electrospun fibers. The study has demonstrated that both drug concentration and scaffolds target topography influenced the scaffolds properties and both can be optimized depending on scaffolds demands.

Animal cells are a complex expression system applied in wide span of biotech and biomedical techniques. Cell growth supporting microenvironment is largely defined by nutrient composition of culture media. Content of the proteins as much as their origin have a key role in preparation of culture media. Fetal bovine serum is still used as a major protein source, despite its compositional inconsistency and safety related risks. To overcome these problems, cell culture experts developed serum- free media where protein hydrolysates, preferably of plant origin, replace serum proteins. Among the most promising substances for the hydrolysate production are protein rich byproducts of kitchen oil industry called oil-cakes or oil-meals. Oil-cakes of hempseed contain about 30% proteins, and as such can be processed into nutrient media supplement for animal cell cultivation. The main objective of the proposed study is to determine the effects of hempseed oil-cake protein hydrolysates on growth and productivity of IgG producing cell lines. The protein hydrolysis was carried out with commercial microbial proteases. Our preliminary results indicated potential application of the hydrolysates as beneficial culture media component.

In this work the function and application of titanium dioxide as a filler in a composite system polycaprolactone/titanium dioxide (PCL/TiO2) was examined. Titanium dioxide was applied by ultrasonic bath on already electrospun PCL fibrous scaffold treated and non-treated with NaOH. A procedure of surface modification of the electrospun PCL fibrous scaffold was made to enhance the interaction of the surface with the TiO2 particles. The surface modification was performed using NaOH for the formation of carboxyl groups on the fibers‘ surfaces. The water contact angle was measured by goniometer to prove the change from hydrophobic to hydrophilic polymer surface. SEM was used to study the morphology structure of the electrospun PCL fibrous scaffold before and after NaOH treatment and introduction of TiO2. The content of TiO2 on the electrospun PCL fibrous scaffold was determined by TGA. After NaOH treatment the surface of the electrospun PCL fibrous scaffolds changed from hydrophobic to hydrophilic. SEM micrographs show that with the sonification of 30 min homogeneous TiO2 particles distribution was obtained, while after sonification of 60 min, the TiO2 particles tend to agglomerate. The modification of the scaffold surface with NaOH enhances the adhesion of the TiO2 filler. TG analysis show that longer treatment of the electrospun PCL fibrous scaffolds in the ultrasonic bath gives lower thermal stability. The time of 30 minutes in the ultrasonic bath is optimal to provide sufficient amount of the TiO2 particles on the electrospun PCL fibrous scaffold.

In this work we describe the adaptation of channel catfish ovary (CCO) cell line to commercially available Ultra Culture serum-free medium by gradual reduction of serum concentration from 10 to 0 %. With this approach we obtained CCO cells fully adapted to serum-free conditions in 32 days. Growth, nutritional and morphological characteristics of these cells remained unchanged when compared to the control group kept in the presence of serum. Additionally, three commercially available protein hydrolysates were tested for the effects on growth performance of the newly serum-free adapted CCO cells. Supplementation with wheat gluten hydrolysate resulted in growth similar to serum free medium solely, while yeast and soy hydrolysates showed inhibitory effects on the cell growth. Taken together, the successful adaptation of CCO cells to serum-free conditions indicates their potential to be used in cytotoxicity assays when serum omission is demanded or for developing serum free bioprocesses using CCO cells. However, a more extended study on nutrient supplementation is still required to further boost the cell growth in a serum free culture.

Over the past three decades a growing awareness of environmental protection prompted the development of so-called green and sustainable technologies. Therefore, academic and wider community intensively explores new chemicals and safer, more energy efficient processes based on a rational compromise between economic, social, and environmental requirements. Due to low volatility and stability, ionic liquids emerged as a potential replacement for traditional volatile and harmful organic solvents. Various studies have been carried out to validate the green character of ionic liquids, whereby data published suggest that these compounds, due to their relatively high toxicity and poor biodegradability, could have an extremely negative impact on the environment. This paper presents the current knowledge on the toxicity of ionic liquids, with a special emphasis on the mechanisms by which this group of compounds causes changes in the morphology and physiology of organisms at different organisational levels of the ecosystem., U protekla tri desetljeća sve više raste svijest ljudi o potrebi zaštite okoliša, pa se velika pozornost pridaje tzv. zelenim i održivim tehnologijama. Stoga se u akademskim sredinama, a i u široj zajednici, intenzivno istražuju nove kemikalije te sigurniji i energetski učinkovitiji procesi koji se zasnivaju na prihvatljivom kompromisu između ekonomskih, socijalnih i ekoloških zahtjeva. Ionske se kapljevine zbog neznatne hlapljivosti i stabilnosti smatraju potencijalnom zelenom zamjenom za tradicionalna i škodljiva organska otapala. Kako bi se potvrdio zeleni karakter ionskih kapljevina, posljednjih godina provode se različita istraživanja njihova učinka na okoliš. Do sada objavljeni podaci upućuju na to da bi ova skupina spojeva, zbog relativno visoke toksičnosti i slabe biorazgradljivosti, ipak mogla imati izrazito negativan utjecaj na okoliš. U ovom radu prikazana su dosadašnja saznanja o toksičnosti ionskih kapljevina, s naglaskom na mehanizme kojima ova skupina spojeva uzrokuje promjene u morfologiji i fiziologiji organizama koji se nalaze na različitim organizacijskim razinama ekosustava.

Over the past three decades a growing awareness of environmental protection prompted the development of so-called green and sustainable technologies. Therefore, academic and wider community intensively explores new chemicals and safer, more energy efficient processes based on a rational compromise between economic, social, and environmental requirements. Due to low volatility and stability, ionic liquids emerged as a potential replacement for traditional volatile and harmful organic solvents. Various studies have been carried out to validate the green character of ionic liquids, whereby data published suggest that these compounds, due to their relatively high toxicity and poor biodegradability, could have an extremely negative impact on the environment. This paper presents the current knowledge on the toxicity of ionic liquids, with a special emphasis on the mechanisms by which this group of compounds causes changes in the morphology and physiology of organisms at different organisational levels of the ecosystem.

Cytotoxic Effects of Imidazolium Ionic Liquids on Fish and Human Cell LinesIonic liquids bring a promise of a wide range of "green" applications that could replace conventional volatile solvents. However, before these applications become large-scale, their toxicity needs to be investigated in order to predict the impact on human health and environment. In this study we assessed the cytotoxicity of imidazolium ionic liquids (in the concentrations between 0.1 mmol L-1 and 10 mmol L-1) in the ovarian fish cell line CCO and the human tumour cell line HeLa using the MTT cell viability assay. Our results showed that the most cytotoxic ionic liquid was 1-n-butyl-3-methylimidazolium bis(trifluoro methylsulphonyl)imide, [BMIM][Tf2N], followed by 1-n-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF4], 1-n-butyl-3-methylimidazolium hexafluorophosphate [BMIM][PF6], and 1,3-dimethylimidazolium hexafluorophosphate [MMIM][PF6]. Generally, the effects were concentration-dependent. They also depended on the type of anion and the n-alkyl chain length. The comparison between the fish CCO and human HeLa cell lines suggests that CCO cells provide a good biological system for initial toxicity testing of ionic liquids that could replace in vivo bioassays.

In this study the effects of ammonium and lactate on a culture of channel catfish ovary (CCO) cells were examined. We also made investigation on the influence of glutamine, since our previous research revealed that this amino acid stimulated CCO cell growth more than glucose in a concentration-dependent manner. The effect of ammonium in cell culture included the considerable decrease in cell growth rate with eventual growth arrest as well as the retardation of glucose consumption. At ammonium concentrations above 2.5 mM, the cells displayed specific morphological changes. The effect of lactate was different to that of ammonium since the cell growth rate was progressively decreasing with the increase of lactate concentration, whereas the glucose consumption rate remained almost unchanged. Besides that, it was found that lactate was steadily eliminated from the culture medium when its initial concentration was relatively high. The influence of glutamine on CCO cell propagation showed that nutrient requirements of this cell line were mainly dependent on glutamine rather than glucose. The increase in glutamine concentration led to the increase in cell growth rate and consequent ammonia accumulation while the glucose utilization and lactate production were reduced. Without glutamine in culture medium cell growth was arrested. However, the lack of glucose reversed the stimulating effect of glutamine by decreasing cell growth rate and affecting amino acid utilization.

The growth characteristics and influence of glucose and glutamine on the growth and maintenance of channel catfish ovary (CCO) cells were investigated. Besides glutamine, amino acids threonine, arginine, methionine and serine were found to be essential for CCO cell growth. In the glucose-free medium, glutamine is utilized as energy source and no cell growth limitation was observed. However, the lack of glutamine in culture medium did not stimulate CCO cells to efficient glucose consumption. When both glucose and glutamine were deficient, cell growth was also observed suggesting no rigorous nutritional requirements. Obtained results are useful for further understanding of culture processes using CCO cells.

The aim of this study was to determine the toxic effect of atrazine at the ovarian cellular level. Chinese Hamster Ovary (CHO-K1) cell line was used to evaluate the degree of in vitro atrazine cytotoxicity and the morphological changes were followed during the cell death. Application of four bioassays confirmed that atrazine decreases ovarian cell proliferation and IC(50) were determined with each assay after 72 h of exposure. The level of apoptosis in atrazine treated cells was low.

Expression of recombinant proteins exerts stress on cell culture systems, affecting the expression of endogenous proteins, and contributing to the depletion of nutrients and accumulation of waste metabolites. In this work, 2D-DIGE proteomics was employed to analyze differential expression of proteins following stable transfection of a Chinese Hamster Ovary (CHO) cell line to constitutively express a heavy-chain monoclonal antibody. Thirty-four proteins of significant differential expression were identified and cross-referenced with cellular functions and metabolic pathways to identify points of cell stress. Subsequently, 1D-(1)H NMR metabolomics experiments analyzed cultures to observe nutrient depletion and waste metabolite accumulations to further examine these cell stresses and pathways. From among fifty metabolites tracked in time-course, eight were observed to be completely depleted from the production media, including: glucose, glutamine, proline, serine, cystine, asparagine, choline, and hypoxanthine, while twenty-three excreted metabolites were also observed to accumulate. The differentially expressed proteins, as well as the nutrient depletion and accumulation of these metabolites corresponded with upregulated pathways and cell systems related to anaplerotic TCA-replenishment, NADH/NADPH replenishment, tetrahydrofolate cycle C1 cofactor conversions, limitations to lipid synthesis, and redox modulation. A nutrient cocktail was assembled to improve the growth medium and alleviate these cell stresses to achieve a ∼75% improvement to peak cell densities.

With the advent of ionic liquids, much was expected concerning their applicability as an alternative to organic solvents in the chemical technology and biotechnology fields. However, the most studied and commonly used ionic liquids based on imidazolium and pyridinium were found not to be as environmentally friendly as it was first expected. Therefore, a new generation of alternative solvents named natural ionic liquids and deep eutectic solvents, composed of natural and/or renewable compounds, have come into focus in recent years. Since the number of newly synthesized chemicals increases yearly, simple and reliable methods for their ecotoxicological assessment are necessary. Permanent fish cell lines can serve as a test system for the evaluation of a chemical’s cytotoxicity. This paper presents research results on the cytotoxic effects on Channel Catfish Ovary (CCO) cell line induced by fifteen cholinium-based ionic liquids and deep eutectic solvents. Based on the decrease in cell viability, the most obvious toxic effect on CCO cells was caused by ionic liquid choline oxalate, while other solvents tested exhibited low cytotoxicity. Therefore, we can conclude that cholinium-based ionic liquids and deep eutectic solvents are comparatively less toxic to CCO cells than conventional ionic liquids.

Introduction: Delivery of nucleic acid-based molecules in human cells is a highly studied approach for the treatment of several disorders including monogenic diseases and cancers. Non-viral vectors for DNA and RNA transfer, although in general less efficient than virus-based systems, are particularly well adapted mostly due to the absence of biosafety concerns. Non-viral methods could be classified in two main groups: physical and vector-assisted delivery systems. Both groups comprise several different methods, none of them universally applicable. The choice of the optimal method depends on the predefined objectives and the features of targeted micro-environment. Areas covered: In this review, the authors discuss non-viral techniques and present recent therapeutic achievements in ex vivo and in vivo nucleic acid delivery by most commonly used techniques while emphasizing the role of ‘biological particles’, namely peptide transduction domains, virus like particles, gesicles and exosomes. Expert opinion: The number of available non-viral transfection techniques used for human therapy increased rapidly, followed by still moderate success in efficacy. The prospects are to be found in design of multifunctional hybrid systems that reflect the viral efficiency. In this respect, biological particles are very promising.

Expression of recombinant proteins exerts stress on cell culture systems, affecting the expression ofendogenous proteins, and contributing to the depletion of nutrients and accumulation of waste metabo-lites. In this work, 2D-DIGE proteomics was employed to analyze differential expression of proteinsfollowing stable transfection of a Chinese Hamster Ovary (CHO) cell line to constitutively express a heavy-chain monoclonal antibody. Thirty-four proteins of significant differential expression were identifiedand cross-referenced with cellular functions and metabolic pathways to identify points of cell stress.Subsequently, 1D-1H NMR metabolomics experiments analyzed cultures to observe nutrient deple-tion and waste metabolite accumulations to further examine these cell stresses and pathways. Fromamong fifty metabolites tracked in time-course, eight were observed to be completely depleted from theproduction media, including: glucose, glutamine, proline, serine, cystine, asparagine, choline, and hypox-anthine, while twenty-three excreted metabolites were also observed to accumulate. The differentiallyexpressed proteins, as well as the nutrient depletion and accumulation of these metabolites correspondedwith upregulated pathways and cell systems related to anaplerotic TCA-replenishment, NADH/NADPHreplenishment, tetrahydrofolate cycle C1 cofactor conversions, limitations to lipid synthesis, and redoxmodulation. A nutrient cocktail was assembled to improve the growth medium and alleviate these cellstresses to achieve a ∼75% improvement to peak cell densities.

By applying concept and principles of green chemistry into different technological processes, green technologies are developed. The environmental and economic benefits of “green” approach is achieved through several directions, such as the use of renewable raw materials, creation of economic efficiency, the use of alternative reaction conditions, as well as the application of non-conventional solvents. From the point view of green chemistry, alternative solvents, in order to be a “green“ substitution to hazardous organic solvents, should be: non-volatile, non-flammable, stabile, synthesized by an environmentally friendly procedure, nontoxic and biodegradable. The toxic impact of all newly synthesized chemicals, such as alternative solvents, could be determined by methods and techniques of ecotoxicology. Ecotoxicology, an interdisciplinary scientific field, can serve as a way of monitoring the greenness of the processes. In vivo and in vitro experiments are used to study the effects of chemicals on different levels of organizations, from molecules to communities and ecosystem. The usage of in vitro methods is encouraged by a scientific community and regulatory agencies as an alternative to in vivo studies in order to reduce the number of laboratory animals used in the toxicological studies. Therefore, in this paper we gave a brief overview on the usage of animal cell cultures within the field of green chemistry and technology.

Gene delivery methods are essential to understand fundamental cellular mechanisms and to develop new therapies. Recombinant viruses are efficient to transfer nucleic acids but their safety is a concern. In addition, some commercial transfection reagents (including lipids and cationic polymers) and electroporation methods are cytotoxic. Interestingly, the sole expression of G envelope protein of the vesicular stomatitis virus (VSV) in mammalian cells can lead to the formation of VSV-G pseudotyped vesicles (V-VSV-G). In presence of polybrene, these vesicles are able to transfer plasmids in a large panel of animal cells. Unfortunately, the production of V-VSV-G and their use for nucleic acid delivery is poorly documented. Here we propose to improve this promising method of transfection. At first we developed a V-VSV-G production process by transient transfection of HEK-293 cells using polyethylenimine (PEI). Three modes of production were compared: cells cultivated in adherence, in suspension and on micro-carriers. Also we demonstrated that the quantity of vesicles produced depends on the VSV-G sequence used. The harvest of V-VSV-G from cell culture media was also optimized. Then, several parameters potentially involved in the formation and the transfer efficiency of V-VSV-G/DNA complex were studied: polybrene concentration, order of addition of mix transfection components, incubation time of the complexes, medium of transfection, etc. Stability studies also demonstrated that V-VSV-G are robust particles: DNA transfer capacity of V-VSV-G is efficient after 10 freezing and thawing cycles and V-VSV-G can be stored for long term at +4 °C, -20 °C and -80 °C. Finally, V-VSV-G/DNA ratio was optimized for three different cell types. Transfection efficiency of 70 % and 55 % were obtained for HEK-293 and HeLa cells respectively, with 1 µg of V-VSV-G and 0.4 µg of DNA. Transfection of refractory cells such as human myoblasts, reached 25 % with 5 µg of V-VSV-G and 0.8 µg of DNA. V-VSV-G can also deliver large plasmids (18 kb). Furthermore, no cytotoxicity was observed in cells transfected with these complexes. Presently, the potential of V-VSV-G to transfer siRNA is investigated. In conclusion, V-VSV-G is a powerful tool for nucleic acid delivery which could be useful for several applications oriented toward cell and gene therapies.

U ovom radu ispitan je uzgoj stanične linije ovarija kanalskog soma (CCO) na mikronosačima tipa Cytodex1 s ciljem proizvodnje biomase te određivanja uvjeta uzgoja u Cell-spin bioreaktoru. Tijekom uzgoja od 312 h praćena je dinamika prihvaćanja CCO stanica na mikronosače, broj stanica i utrošak glukoze u dva medija s 5% i 2% seruma. Uzgoj CCO stanica u mediju sa smanjenim udjelom seruma rezultirao je većim prinosom stanica i specifičnom brzinom rasta u odnosu na preporučeni medij za uzgoj. Dobiveni rezultati ukazuju da ispitani UltraCulture medij predstavlja dobar odabir u slučaju kad je potrebno vršiti uzgoj CCO stanica u uvjetima smanjenog udjela seruma., In this study Cytodex 1 microcarriers were used for Channel Catfish Ovary (CCO) cell cultivation and biomass production in Spinner-flask. Cell attachment, cell number and glucose consumption were determined during 312 h cultivation in two media with 5% and 2% of serum. CCO cell cultivation in medium with lower serum concentration resulted in higher cell yield and specific growth rate when compared to standard growth medium. Obtained results showed that tested UltraCulture medium was good choice when cultivation of CCO cells in reduced serum conditions is demanded.

Camelid single domain antibodies fused to noncamelid Fc regions, also called chimeric heavy chain antibodies (cHCAb), offer great potential as therapeutic and diagnostic candidates due to their relatively small size (80 kDa) and intact Fc. In this chapter, we describe two approaches, limiting dilution and minipools, for generating nonamplified Chinese hamster ovary cell lines stably expressing cHCAb in suspension and serum-free cultures using a stringent antibiotic selection. Neither of the protocols necessitates the acquisition or implementation of expensive automated infrastructures and thus could be applied in any lab with minimal cell culture setup. The given protocol allows the isolation of stable clones capable of generating up to 100 mg/L of antibody in batch mode performed in shaker flasks.

Ionic liquids bring a promise of a wide range of “green” applications that could replace conventional volatile solvents. However, before these applications become large-scale, their toxicity needs to be investigated in order to predict the impact on human health and environment. In this study we assessed the cytotoxicity of imidazolium ionic liquids (in the concentrations between 0.1 mmol L-1 and 10 mmol L-1) in the ovarian fi sh cell line CCO and the human tumour cell line HeLa using the MTT cell viability assay. Our results showed that the most cytotoxic ionic liquid was 1-n-butyl-3-methylimidazolium bis(trifluoro methylsulphonyl)imide, [BMIM][Tf2N], followed by 1-n-butyl-3-methylimidazolium tetrafl uoroborate [BMIM][BF4], 1-n-butyl-3-methylimidazolium hexafluorophosphate [BMIM][PF6], and 1,3-dimethylimidazolium hexafl uorophosphate [MMIM][PF6]. Generally, the effects were concentrationdependent. They also depended on the type of anion and the n-alkyl chain length. The comparison between the fish CCO and human HeLa cell lines suggests that CCO cells provide a good biological system for initial toxicity testing of ionic liquids that could replace in vivo bioassays., S obzirom na širok raspon primjena ionskih tekućina, prethodno je potrebno ispitati njihovu toksičnost i mogući utjecaj na zdravlje ljudi i okoliš. U ovom radu ispitana je citotoksičnost odabranih imidazolijevih ionskih tekućina na stanicama ovarija riba CCO i ljudskoj tumorskoj staničnoj liniji HeLa primjenom MTT-metode. Izlaganje stanica različitim koncentracijama ionskih tekućina [(0,1 do 10) mmol L-1] rezultiralo je uglavnom citotoksičnim učincima ovisnima o koncentracijama ionske tekućine. Na temelju dobivenih vrijednosti EC50 najtoksičnija ionska tekućina je 1-n-butil-3-metilimidazolijev bis(trifl uorome tilsulfonil)imid [BMIM][Tf2N], zatim 1-n-butil-3-metilimidazolijev tetrafl uoroborat [BMIM][BF4],1-nbutil- 3-metilimidazolijev heksafluorofosfat [BMIM][PF6] i 1,3-dimetilimidazolijev heksafluorofosfat [MMIM][PF6]. Općenito, toksičnost je bila ovisna o koncentraciji, tipu aniona i duljini n-alkilnog lanca. Nakon usporedbe rezultata toksičnosti na CCO i HeLa-stanicama smatramo da CCO-stanice mogu biti dobar biološki sustav za početna ispitivanja toksičnosti ionskih tekućina u cilju zamjene in vivo testova na ribama.

Camelid single domain antibodies fused to noncamelid Fc regions, also called chimeric heavy chain antibodies (cHCAb), offer great potential as therapeutic and diagnostic candidates due to their relatively small size (80 kDa) and intact Fc. In this chapter, we describe two approaches, limiting dilution and minipools, for generating nonamplified Chinese hamster ovary cell lines stably expressing cHCAb in suspension and serum-free cultures using a stringent antibiotic selection. Neither of the protocols necessitates the acquisition or implementation of expensive automated infrastructures and thus could be applied in any lab with minimal cell culture setup. The given protocol allows the isolation of stable clones capable of generating up to 100 mg/L of antibody in batch mode performed in shaker flasks.

There is considerable concern about the substances present in the environment and their potential to interfere with the endocrine system of vertebrates. Among these, the so-called endocrine-disrupting compounds, which can modulate or disrupt developmental and reproductive processes, substances with estrogenic activity have attracted most attention. Concerns about the presence of these compounds in the environment have led to the development of screening and testing assays that are able to detect such substances and evaluate their potential to induce adverse effects. In vitro systems such as mammalian and fish cell lines have become of growing importance in toxicity testing of such compounds. The cytotoxic and apoptotic effects induced by 17α-ethynylestradiol and diethylstilbestrol were studied on CHO-K1 cell line. Trypan blue exclusion method was used to determine the cell viability. Cytotoxicity of 17α-ethynylestradiol (0.34–34 μM) and diethylstilbestrol (0.37–37 μM) was found to be concentration-dependent with IC50 values of 12.8 and 10.4 μM after 72 h of exposure, respectively. In treated CHO-K1 culture cell death was assessed by determining morphological changes by haematoxilyn and eosin staining, nuclear morphology by fluorescein diacetate/propidium iodide staining and fluorescence microscopy, DNA fragmentation by TUNEL method and translocation of phosphatidyl serine by flow cytometry. The obtained results showed that 17α-ethynylestradiol induced apoptosis, while diethylstilbestrol induced necrosis in the treated CHO-K1 cells., Javnost sve više zabrinjavaju brojne tvari u okolišu koje mogu utjecati na endokrini sustav kralježnjaka. Među tzv. endokrinim modulatorima, koji mogu poremetiti ili potpuno narušiti razvojne i reproduktivne procese u organizmu, najveću pozornost privlače tvari što imaju estrogenu aktivnost. Ta je zabrinutost potaknula razvoj novih metoda za njihovo praćenje i određivanje te izbjegavanje neželjenih učinaka. Testovi in vitro sa staničnim linijama sisavaca i riba postali su važni modelni sustavi u ispitivanju toksičnosti tih spojeva. Citotoksični i apoptotički učinci 17α-etinilestradiola i dietilstilbestrola ispitani su na CHO-K1 staničnoj liniji. Za određivanje preživljavanja stanica upotrebljena je metoda tripan plavo. Citotoksičnost 17α-etinilestradiola (0,34-34 µM) i dietilstilbestrola (0,37-37 µM) ovisi o koncentraciji, a izračunate IC50 vrijednosti nakon 72 sata nakon tretmana iznosile su pojedinačno 12,8 i 10,4 µM. Smrt CHO-K1 stanica praćena je na temelju uočenih morfoloških promjena, bojenjem hematoksilinom i eozinom, zatim promjena u izgledu jezgre, bojenjem fluorescein diacetatom/propidijevim jodidom i fluorescentnom mikroskopijom, te prema fragmentaciji DNA TUNEL metodom i translokaciji fosfatidilserina protočnom citometrijom. Dobiveni rezultati upućuju na to da 17α-etinilestradiol uzrokuje apoptozu u CHO-K1 stanicama, a dietilstilbestrol njihovo odumiranje procesom nekroze.

The effects of synthetic estrogens 17α-ethynylestradiol (EE2) and diethylstilbestrol (DES) were compared on cell proliferation and morphology in Channel Catfish Ovary (CCO) and Chinese Hamster Ovary (CHO-K1) cells. EE2 exposure (0.1 and 0.5 μg/mL) induced stimulatory effect on CCO and CHO-K1 cell proliferation, while higher concentrations (1-10 μg/mL) showed cytotoxic effects. Increase in DES concentrations mainly resulted in dose-depended increase in cytotoxicity in both cell lines. Morphological changes induced by EE2 and DES exposure (5 μg/mL) showed disrupted cell monolayer and increased number and size of lysosomes. Comparison of IC(50) values showed almost equal sensitivity towards cytotoxicity of tested compounds in CCO and CHO-K1 cells.

Growth factors from neural tissues have been described as potent mitogens for a wide variety of mesoderm- and ectoderm-derived cells in vitro. We used porcine brain extract for in vitro testing of proliferation properties on primary ovarian cells, uterine cells, and cardiomyocytes in culture as well as for BHK-21 [C-13] cell line. The addition of this extract accelerates proliferation in all examined cultures. It also lowers serum requirement and shortens the cultivation period for BHK-21 [C-13] cells. Fibroblast growth factors from brain of different species, but not porcine, are already characterized and their proliferative effect proved. Therefore, we purified, determined, and confirmed the presence of basic fibroblast growth factor in porcine brain extract by Western blot analysis and showed its biological activity on BHK-21 [C-13] cells.

A novel, disposable-bag bioreactor system that uses wave action for mixing and transferring oxygen was evaluated for BHK 21 C13 cell line growth and Aujeszky's disease virus (ADV) production. Growth kinetics of BHK 21 C13 cells in the wave bioreactor during 3-day period were determined. At the end of the 3-day culture period and cell density of 1.82x10(6) cells ml-1, the reactor was inoculated with 9 ml of gE- Bartha K-61 strain ADV suspension (10(5.9) TCID50) with multiplicity of infection (MOI) of 0.01. After a 144 h incubation period, 400 ml of ADV harvest was obtained with titre of 10(7.0) TCID 50 ml-1, which corresponds to 40,000 doses of vaccine against AD. In conclusion, the results obtained with the wave bioreactor using BHK 21 C13 cells showed that this system can be considered as suitable for ADV or BHK 21 C13 cell biomass production.

The cultivation of animal cells is of great importance for effective and reasonable bioprocess production of various therapeutic proteins as well as for fundamental laboratory research. The metabolic patterns of animal cells are altered substantially during the actions of establishment of a typical cell line designed for in vitro techniques. Therefore, characterization and understanding of cell metabolism are needed to improve the processes with cultivated animal cells. The channel catfish ovary cell line (CCO) was derived from the ovaries of a juvenile channel catfish some thirty years ago. Since then, it has been used for channel catfish virus vaccine production and related in vitro laboratory research. However there is still no enough data for basal understanding of their metabolism. We report on growth characteristics of CCO cells cultivated in multiwell plates and Wave bioreactor. Specific growth rate of CCO cells in multiwell plates was 0, 0206 h-1. The concentration of glucose in culture media remained unchanged through whole cultivation period. Glutamine was almost completely depleted and ammonia, its major wasteproduct, reached concentration of 2, 3 mM. Amino acid analysis of culture media showed that highly consumed amino acids, besides glutamine, were arginine, serine, and threonine, suggesting that they were main energy source. Cytodex 1 microcarriers were used in order to investigate medium-scale cultivation parameters of CCO cells in the Wave bioreactor. In this cultivation system oxygen transfer and mixing are accomplished by the wave-induced agitation. Micocarrier confluence was reached with cell density of 1, 8 x 10e6 cells/mL and cell growth rate of 0, 0182 h-1. The results proved the feasibility of employed culture conditions for cultivation of CCO cells.

Depending on the concentration, catecholamines activate various intracellular signaling pathways and can induce apoptosis in cardiac myocytes. Although 5, 5’ , 6, 6’ -tetrachloro-1, 1’ , 3, 3’ -tetraethylbenzimidazolocarbocyanine iodide (JC-1) has been previously used to study mitochondria in intact cardiomyocytes, there have been no reports on the detection of apoptosis in neonatal cardiomyocytes in combination with flow cytometry and confocal microscopy. In our study, neonatal rat cardiomyocytes were exposed to norepinephrine (NE) and isoproterenol (ISO) in concentrations of 1 microM and 10 microM for 48 hours. NE concentrations of 1 microM and 10 microM decreased the number of viable cardiomyocytes by 18% (*p

There is considerable concern about the substances present in the environment and their potential to interfere with the endocrine system of vertebrates. Among these, the so-called endocrine-disrupting compounds, which can modulate or disrupt developmental and reproductive processes, substances with estrogenic activity have attracted most attention. Concerns about the presence of these compounds in the environment have led to the development of screening and testing assays that are able to detect such substances and evaluate their potential to induce adverse effects. In vitro systems such as mammalian and fish cell lines have become of growing importance in toxicity testing of such compounds. The cytotoxic and apoptotic effects induced by 17α-ethynylestradiol and diethylstilbestrol were studied on CHO-K1 cell line. Trypan blue exclusion method was used to determine the cell viability. Cytotoxicity of 17α-ethynylestradiol (0.34–34 μM) and diethylstilbestrol (0.37–37 μM) was found to be concentration-dependent with IC50 values of 12.8 and 10.4 μM after 72 h of exposure, respectively. In treated CHO-K1 culture cell death was assessed by determining morphological changes by haematoxilyn and eosin staining, nuclear morphology by fluorescein diacetate/propidium iodide staining and fluorescence microscopy, DNA fragmentation by TUNEL method and translocation of phosphatidyl serine by flow cytometry. The obtained results showed that 17α-ethynylestradiol induced apoptosis, while diethylstilbestrol induced necrosis in the treated CHO-K1 cells.