Chinese hamster ovary cells produce sufficient recombinant insulin-like growth factor I to support growth in serum-free medium - Serum-free growth of IGF-I producing CHO cells

Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture, and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/10(6) cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin.

Adipose tissue mesenchymal stem cell expansion in animal serum-free medium supplemented with autologous human platelet lysate

BACKGROUND: Mesenchymal stem cells (MSCs) have been considered for human regenerative therapy applications, and safe culture and expansion protocols are needed especially in the context of interspecies contamination. Human platelet lysate (PL) has been proposed as animal serum substitute during in vitro MSC expansion. In this work, a simplified and efficient method to obtain autologous PL to replace animal serum in cell culture applications is described. STUDY DESIGN AND METHODS: PL obtained by freezing and centrifugation procedures was tested as medium supplement for human adipose mesenchymal stem cell (hASC) culture. Differential proliferation, immunophenotypic changes, and differentiation under PL or fetal bovine serum (FBS) were assessed. RESULTS: In contrast to 10% FBS supplementation, cell population doubling time was significantly lower when hASCs were cultured with the same concentration of PL ( PL 22.9 +/- 1.5 hr vs. FBS 106.7 +/- 6.5 hr, t test, p < 0.05). Furthermore, hASCs maintained with 2.5% PL supplementation also showed satisfactory results. Immunophenotypic analysis revealed no differences between hASCs cultivated with PL or FBS supplementation and both cultures retained the potential to differentiate into adipose cells. These results demonstrate that autologous PL obtained from the same donor can be used as animal serum substitute in hASC culture. CONCLUSIONS: Taken together, evidence is provided that platelets provided by a single donor are sufficient to obtain PL for hASC propagation for clinical-scale applications mitigating the potential untoward side effects associated with the use of animal-derived reagents.

In vitro susceptibility to antimycotic drug undecanoic acid, a medium-chain fatty acid, is nutrient-dependent in the dermatophyte Trichophyton rubrum

The antimycotic activity of fatty acids has long been known, and their presence in human skin and sweat appears to protect the host against superficial mycoses. Undecanoic acid is a medium-chain fatty acid that has been used in the treatment of dermatophytoses in humans. In this study, we selected one Trichophyton rubrum undecanoic acid-resistant strain that showed a marked reduction in its capacity to grow on human nail fragments, which correlated with the reduced activity of secreted keratinolytic proteases. Moreover, the susceptibility of T. rubrum to undecanoic acid is also dependent on the carbon source utilized by both control and resistant strains. The growth of the control strain was strongly inhibited by undecanoic acid in Sabouraud medium or in cultures supplemented with low-fat milk, whereas it was ineffective when the cultures were supplemented with Tween 20 or keratin as the carbon source, suggesting that nutrient conditions are crucial in establishing a susceptibility to antifungal drugs, which is helpful for the isolation and characterization of resistant strains, and in the screening for new antifungal drugs.

Supplemented tissue culture medium 199 is a better medium for in vitro maturation of oocytes from women with polycystic ovary syndrome women than human tubal fluid

Objective: To compare oocyte maturation, fertilization and cleavage rates, and embryonic developmental quality after culture of human immature oocytes from polycystic ovary syndrome (PCOS) patients in human tubal fluid (HTF) or tissue culture medium (TCM) 199. Design: Prospective, randomized, controlled trial. Setting: University hospital. Patient(s): Thirteen women undergoing 23 in vitro maturation cycles, from whom 119 oocytes were retrieved. Intervention(s): Cumulus-enclosed germinal vesicle-stage oocytes matured in TCM-199-supplemented or HTF-supplemented media. Main Outcome Measure(s): Oocyte maturation and fertilization rates, embryonic developmental quality. Result(s): Significant differences were observed between TCM 199 and HTF regarding maturation rate (82% vs. 56.9%), fertilization rate (70% vs. 39.4%), and embryo quality (81.3% vs. 41.7%). Conclusion(S): Human tubal fluid medium, although widely used for embryo fertilization and maintenance in IVF techniques, is not,in appropriate medium for the maturation of oocytes obtained from PCOS patients in nonstimulated cycles. (Fertil Steril(R) 2009;91:509-13. (C)2009 by American Society for Reproductive Medicine.)

Performance of a chromogenic medium for the isolation of Listeria monocytogenes in food

According to the producers, CHROMagar (TM) Listeria medium comes to facilitate the detection, differentiating Listeria monocytogenes from the other species, directly on the isolation process, allowing final results in 72 h, while the traditional method takes up to 10 days. A total of 120 food samples of sliced cooked ham, ground beef and frankfurters was analyzed. From 151 colonies presenting typical L. monocytogenes characteristics on CHROMagar (TM) Listeria (bluish, surrounded by a white halo), only 95 (62.9%) were confirmed as L. monocytogenes. The medium was highly sensitive to detect L. monocytogenes in ground beef and frankfurter, but not in sliced ham. (c) 2007 Elsevier Ltd. All rights reserved.

Effects of gonadotropin-exposed medium with high concentrations of progesterone and estradiol-17 beta on in vitro maturation of canine oocytes

During the process of maturation in the oviduct, canine oocytes in the germinal vesicle stage are exposed to decreasing levels of estradiol-17 beta and increasing levels of progesterone. However, hormone concentrations in the microenvironments in which they act are higher than serum concentrations. Therefore, the aim of the present study was to compare the meiotic competence of canine oocytes harvested from anestrous bitches in culture medium containing high concentrations (20 mu g ml(-1)) of estradiol-17 beta and/or progesterone in association to gonadotropins (luteinizing hormone and follicle-stimulating hormone) using three different maturation periods (48, 72, and 96 h). Oocytes were cultured in tissue culture medium (TCM-199) and arranged in four experimental groups: group control, group E2 (estradiol-17 beta), group P4 (progesterone), and group E2 + P4. Regardless of the maturation period, groups P4 and E2 + P4 presented statistically higher rate of germinal vesicle breakdown oocytes compared to the group control and group E2. There were no significant differences among groups on germinal vesicle, metaphase I, metaphase II, and degenerated or unidentifiable oocytes rates. The mean percentage of metaphase II oocytes was higher at 96 h when compared to 72 h. Results of the present research indicate no influence of estradiol-17 beta supplementation, unless in association with progesterone. There is an evidence of the positive effect of progesterone on germinal vesicle breakdown. Results also showed that extended periods of in vitro maturation affect positively maturation rates to metaphase II of low competent oocytes harvested from anestrous bitches, independent of the maturation media. In conclusion, high concentrations of steroids, especially progesterone, have positive effect on in vitro oocyte maturation when the oocytes are derived from the anestrous status.

Tooth Replantation After Use of Euro-Coffins Solution or Bovine Milk as Storage Medium: A Histomorphometric Analysis in Dogs

Purpose: Euro-Collins solution was developed for the preservation of organs for transplantation, whose characteristics have raised interest for its use as a storage medium for avulsed teeth before replantation. This study evaluated histologically and morphometrically the healing process of dog teeth replanted after storage in Euro-Collins solution or bovine milk. Materials and Methods: Eighty roots of 4 young adult mongrel clogs were randomly assigned to 4 groups (n = 20) and the root canals were instrumented and obturated with gutta-percha and a calcium hydroxide-based sealer. After 2 weeks, the teeth were extracted and subjected to the following protocols: GI (negative control), replantation immediately after extraction; GII (positive control), bench-drying for 2 hours before replantation; GIII and GIV, immersion in 10 mL of whole bovine milk and Euro-Collins solution at 4 C, respectively, for 8 hours before replantation. The animals were sacrificed 90 days postoperatively. The pieces containing the replanted teeth were subjected to routine processing for histologic and histometric analyses under light microscopy and polarized light microscopy. Results: Root resorption was observed in all groups. GII exhibited the greatest loss of dental structure (P < .01), and inflammatory resorption was predominant in this group. Storage in milk showed poorer results than immediate replantation and storage in Euro-Collins solution (P < .01). The teeth stored in Euro-Collins solution presented similar extension of root resorption and periodontal ligament reorganization to those of immediately replanted teeth. Conclusions: The findings of this study suggest that the Euro-Collins solution is an adequate storage medium for keeping avulsed teeth for up to 8 hours before replantation. Crown Copyright (C) 2010 Published by Elsevier Inc on behalf of American Association of Oral and Maxillofacial Surgeons. All rights reserved. Oral Maxillofac Surg 68:111-119, 2010

Study of the effectiveness of propolis extract as a storage medium for avulsed teeth

The purpose of the present study was to evaluate the efficacy of propolis extract in maintaining the viability of human periodontal ligament (PDL) cells, and to radiographically analyze tooth replantation and the adjacent periodontium in dogs after storage in this extract. Human PDL cells were incubated with the experimental media propolis, milk, saliva, Hank`s balanced salt solution (HBSS), and Dulbecco`s modified Eagles medium (DMEM, positive controls), and distilled water (negative control). Cell viability was determined 0, 1, 3, 6, 12, and 24 h later by colorimetric MTT assay. Thirty incisors from dogs were divided into two storage time blocks (1 and 3 h) and were maintained in the experimental media. HBSS served as a positive control, and dry teeth (on gauze) as a negative control. The replanted teeth were radiographed once per month for 6 months. The radiographic images were standardized by the shortening/lengthening factor, and were both qualitatively and quantitatively analyzed. The in vitro results showed that the efficacy of propolis in maintaining functional viability of PDL cells was similar to that of milk. Propolis and milk were significantly better than controls from the 6-h time period. The in vivo results showed that teeth maintained in propolis medium exhibited replacement resorption with significant reduction in tooth length, similar to teeth maintained in saliva and dried teeth. This resorption was less intense with the 3-h storage time than the 1-h storage time. Conditions close to normal were found in teeth maintained in milk, similar to the HBSS control. Therefore, although propolis was effective in maintaining the viability of human PDL cells, resorption of the tooth replantation in dogs occurred under these experimental conditions.

Near-resonant holographic interferometry of hypersonic flow

Near-Resonant Holographic Interferometry is a powerful technique which extends the established advantages of conventional holographic interferometry by allowing a species-specific number density to be determined. It has been tested in the harsh flow conditions generated in a high enthalpy facility yielding information about the shock shape on a cylindrical body and on the distribution of a trace species seeded into the flow.