Enrichment Of Culture Medium Research Articles

Meiotic and developmental competence of growing pig oocytes derived from small antral follicles is enhanced in culture medium containing FGF2, LIF, and IGF1 (FLI medium)

BackgroundOocytes of large animal species isolated from small ovarian follicles (< 2 mm) are less competent to support early embryonic development after in vitro maturation and fertilization than their counterparts isolated from medium-sized and preovulatory follicles. This study aimed to assess the effect of a new maturation medium containing FGF2, LIF, and IGF1 (FLI medium) on the meiotic and developmental competence of pig cumulus-oocytes complexes (COCs) derived from the small and medium-sized follicles.MethodsThe growing oocytes were isolated from 1 to 2 (small follicle; SF) and the fully-grown ones from 3 to 6 (large follicle; LF) mm follicles and matured in a control M199 medium with gonadotropins and EGF and the FLI medium enriched by the triplet of growth factors. The matured oocytes were parthenogenetically activated and cultured to the blastocyst stage. Chromatin configuration before and during the culture and MAP kinase activity were assessed in the oocytes. Finally, the expression of cumulus cell genes previously identified as markers of oocyte quality was assessed.ResultsThe maturation and blastocyst rates of oocytes gained from LF were significantly higher than that from SF in the control medium. In contrast, similar proportions of oocytes from LF and SF completed meiosis and developed to blastocysts when cultured in FLI. Most of the oocytes freshly isolated from SF possessed germinal vesicles with fine filaments of chromatin (GV0) or chromatin surrounding the nucleolus (GVI; 30%); the oocytes from LF were mainly in GVI (or GVII) exhibiting a few small lumps of chromatin beneath the nuclear membrane. When cultured in the FLI medium for 16 h, an acceleration of the course of maturation in oocytes both from SF and LF compared to the control medium was observed and a remarkable synchrony in the course of chromatin remodeling was noticed in oocytes from SF and LF.ConclusionsThis work demonstrates that the enrichment of culture medium by FGF2, LIF, and IGF1 can enhance the meiotic and developmental competence of not only fully-grown, but also growing pig oocytes and significantly thus expanding the number of oocytes available for various assisted reproductive technology applications.

A 100-fold increasing in measles virus titer following enrichment of culture medium with MgSO4

Measles is a contagious disease which causes more than 140,000 deaths per year. The aim of this research was to increase the titer of harvested Measles virus during its propagation in cell culture. For this purpose, the effect of Magnesium Sulfate on titer of Measles virus was evaluated. Specific concentrations of magnesium sulfate salt along with measles virus of AIK-C strain (vaccine strain) were added into chick embryos cell culture medium. Then at the specific times interval the virus concentration in the control and test samples were determined by CCID50 method. Finally the obtained results were compared by Real Time _RT PCR method. The results showed that, MgSo4 increases the titer of Measles virus in the harvested suspensions. The mean increase in viral titer in comparison to the control was determined as 2.3 Log (in macrotitration method) and 2.17 Log (in microtitration method). Also there were significant correlation between CCID50 method and Real Time _RT PCR. In this research, the titer of harvested Measles virus was increased by 100 fold. This method making a significant increase in efficacy of the virus propagation process, so, the expenses of vaccine production will be decreased.

Dietary supplements based on selenium containing culture of lactic acid bacteria

The article describes the role of selenium in the humankind being. The analysis based on the published data shows that the biological synthesis is a perspective way to obtain an organic form of selenium, which can be used in dietary supplements. The ability of lactic acid bacteria (Lactobacillus acidophilus 412/307)to accumulate inorganic forms of selenium (selenites, selenates), turning them into organic forms, with purposeful trace element enrichment of culture medium is described in the article. The main organic forms of selenium, which are being used in the process of biotransformation from its inorganic forms by microorganisms, have been reorganized. The relationship between the increasing of concentrations of sodium selenite in the culture medium and the growth of biomass of lactic acid bacteria was established. It was found the depressing effect of increasing concentrations of sodium selenite on optical density rate. It was estabilished the optimal conditions for the maximum accumulation of selenium containing culture of lactic acid bacteria. The influence of selenium concentration on the lactic acid bacteria biomass accumulation was determined also by changing the values of optical density. Due to the obtained data, the selenium containing dietary supplement «Selenolakt» was created. The main microbiological indicators that characterize the quality of the obtained product are given. The content of organic form of selenium in products reaches –195 ± 1 mkg/g.

Crocetin improves the quality of in vitro–produced bovine embryos: Implications for blastocyst development, cryotolerance, and apoptosis

The aim of this work was to assess the effect of supplementation of bovine culture medium with the natural antioxidant crocetin on in vitro blastocyst development and quality. This was evaluated as cryotolerance, apoptosis index, and total cells number and allocation. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, presumptive zygotes were cultured in synthetic oviduct fluid medium, supplemented with 0, 1, 2.5, and 5 μM crocetin (experiment 1) at 39 °C under humidified air with 5% CO2, 7% O2, and 88% N2. On Day 7, embryo yields were assessed and the blastocysts were vitrified by Cryotop method in 16.5% ethylene glycol, 16.5% DMSO, and 0.5 M sucrose. Finally, blastocysts produced on Day 8 in the absence (control) and presence of 1 μM crocetin were used for terminal deoxynucleotidyl transferase–mediated dUTP nick end labelling and differential staining to evaluate, respectively, the apoptotic rate and the allocation of cells into inner cell mass (ICM) and trophectoderm (TE) lineages (experiment 2). Embryo development was higher in the 1 μM crocetin group compared to the control, both in terms of total embryo output (37.7 ± 4.2%, 52.9 ± 6.3%, 40.9 ± 7.6%, and 42.4 ± 8.7%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.01) and grade 1 and 2 blastocysts (33.6 ± 4.9%, 46.1 ± 7.3%, 37.8 ± 7.9%, and 39.4 ± 7.9%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.05). Moreover, the percentage of fast-developing embryos increased in 1 μM crocetin group compared to the control (23.4 ± 4.7%, 32.7 ± 6.6%, 27.2 ± 6.6%, and 30.1 ± 7.2%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.05). In addition, the enrichment of culture medium with 1 μM crocetin improved embryo cryotolerance compared to the control, as indicated by higher hatching rates recorded after 48 hours postwarming culture (46.5% vs. 60.4%; P < 0.05). Furthermore, 1 μM crocetin decreased both the average number (9.9 ± 0.4 vs. 7.1 ± 0.3) and the percentage of apoptotic cells (7.1 ± 0.4 vs. 4.2 ± 0.2) in blastocysts compared to the control (P < 0.01). However, no differences were recorded in the average number of ICM, TE, and total cells between 1 μM crocetin and control groups. In conclusion, the enrichment of bovine culture medium with 1 μM crocetin increased both blastocyst yield and quality, as indicated by the improved chronology of embryo development, increased resistance to cryopreservation, and reduced incidence of apoptosis.

Human cell lines: A promising alternative for recombinant FIX production

Factor IX (FIX) is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the Hemophilia B treatment. We evaluated the potential of recombinant human FIX (rhFIX) expression in 293T and SK-Hep-1 human cell lines. SK-Hep-1-FIX cells produced higher levels of biologically active protein. The growth profile of 293T-FIX cells was not influenced by lentiviral integration number into the cellular genome. SK-Hep-1-FIX cells showed a significantly lower growth rate than SK-Hep-1 cells. γ-carboxylation process is significant to FIX biological activity, thus we performed a expression analysis of genes involved in this process. The 293T gene expression suggests that this cell line could efficiently carboxylate FIX, however only 28% of the total secreted protein is active. SK-Hep-1 cells did not express high amounts of VKORC1 and carboxylase, but this cell line secreted large amounts of active protein. Enrichment of culture medium with Ca+2 and Mg+2 ions did not affect positively rhFIX expression in SK-Hep-1 cells. In 293T cells, the addition of 0.5 mM Ca+2 and 1 mM Mg+2 resulted in higher rhFIX concentration. SK-Hep-1 cell line proved to be very effective in rhFIX production, and it can be used as a novel biotechnological platform for the production of recombinant proteins.

Ability of rumen protozoa Diploplastron affine to utilize β-glucans

The ability of the rumen ciliates to utilize β-glucans other than cellulose and xylan is currently being recognized. The objective of the present study was to characterize the ability of the ciliate Diploplastron affine to digest some pachyman, laminarin, pustulan, curdlan and lichean. The protozoa were isolated from the rumen of sheep and either grown in vitro or inoculated into the rumen of ciliate-free sheep and maintained in natural conditions. In vitro culture studies showed that the enrichment of culture medium with the examined saccharides results in an increase in the number of ciliates in comparison to the control cultures. The increase was over 36 and 15 % when the growth medium was supplemented with pachyman (1,3-β-glucan) and pustulan (1,6-β-glucan), respectively. A positive correlation was also found between the population density of ciliates and the dose of saccharide supplemented to the growth medium. Enzyme studies were performed using the crude enzyme preparation obtained from ciliates treated with antibiotics. The ability of ciliates to digest the examined β-glucans was tested by the quantification of reducing sugars released from the mentioned substrates during the incubation with crude enzyme preparation. The results showed that D. affine ciliates were able to digest both of them. The mean degradation rate varied between 6.7 and 28.2 μmol/L glucose per mg protein per h for pustulan and lichean, respectively, whereas the digestion velocity was the highest at 5.0–5.5 pH and 45–50°C.

Selective polyunsaturated fatty acids enrichment in phospholipids from neuronal-derived cell lines

Most studies aimed at exploring the molecular and cellular properties of plasma membranes in neural tissues make use of cell lines. However, cell membrane lipid composition of cell lines is notably different from that of brain tissues where they presumably derive from. Using septal-derived SN56 cells and hippocampal-derived HT22 cells, we demonstrated that cell lines exhibit lower contents of saturated (18:0) and long polyunsaturated fatty acids (PUFA; 20:4 n − 6 and especially 22:6 n − 3), as well as higher monounsaturated fatty acid contents (mainly 18:1 n − 9), compared to mouse brain. Also, cell lines exhibited higher contents of sterol esters and lower contents of cholesterol and phospholipids, especially phosphatidylethanolamine and phosphatidylserine. We have also evaluated the effects of different ( n − 3/ n − 6) PUFA enrichments on fatty acid and phospholipid contents in these cell lines. Our results show that enrichment of culture medium with 22:6 n − 3 and 20:4 n − 6 in a 70/30 proportion during 48 h, using fat-free bovine serum albumin as vehicle, successfully readjusted fatty acid profiles in cell line-polar lipids to values found in natural nerve cells. Interestingly, no differences in cell survival were observed upon enrichment. The generalization of these methodologies would allow a more feasible adaptation of cellular models to the study of in vivo nerve physiology.