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Nishii, R., K. Imai, H. Koyama, and O. Dochi. "166 EFFECT OF INDIVIDUAL CULTURE SYSTEM ON IN VITRO DEVELOPMENT OF IN VITRO-MATURED - IN VITRO-FERTILIZED BOVINE EMBRYOS." Reproduction, Fertility and Development 24, no. 1 (2012): 195. http://dx.doi.org/10.1071/rdv24n1ab166.
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An individual in vitro culture system for bovine embryo needs to be developed for the study of embryo developmental competence. The objective of this study was to examine the effect of individual culture systems on the development of in vitro-matured–in vitro-fertilized bovine embryos. Two individual culture systems were compared. Cumulus–oocyte complexes (COC) were collected by aspiration of ovarian follicles (diameter, 2 to 5 mm) obtained from a local abattoir. The COC were matured in TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 of FSH. Groups of 20 COC were incubated in 100-μL droplets of IVM media at 38.5°C under an atmosphere of 5% CO2 for 20 h. After 18 h of gamete co-culture (3 × 106 sperm mL–1), the presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum at 38.5°C under an atmosphere of 5% CO2, 5% O2 and 90% N2 for 9 days (fertilization = Day 0). The presumptive zygotes were randomly assigned to 1 of the following 3 treatments: single culture (1 zygote was cultured in a 5-μL droplet), well-of-the-well (WOW; Sugimura et al. 2010 Biol. Reprod. 83, 970–978) culture (25 zygotes were cultured individually in each 125-μL droplet) and control culture (25 zygotes were cultured in a 125-μL droplet). Embryo development was evaluated for cleavage and blastocyst rates, on Day 2 and Day 7 to 9 after IVF, respectively. The rates of development up to the blastocyst stage and total cell number in the blastocysts, determined by an air-drying method, were investigated. The cleavage and blastocyst rates were analysed by the chi-square test and the total cell numbers were analysed by ANOVA. The cleavage rates were significantly higher in the control and WOW groups than in the single-culture group (P < 0.01) and the blastocyst rates were significantly lower in the single-culture group than in the control culture group (P < 0.05; Table 1). The total cell numbers (mean ± s.d.) of blastocysts did not significantly differ between the single culture (154.6 ± 21.8), control culture (155.2 ± 22.5) and WOW culture (159.8 ± 27.0) groups. These results indicate that although the blastocyst rate was lower in the single-culture system than in the WOW or group culture system, in vitro-matured–in vitro-fertilized bovine embryos can be cultured using the single-culture system. Moreover, the quality of blastocysts developed by the single-culture system is the same as that of blastocysts developed using the other 2 culture systems. Table 1.Effect of different culture methods for bovine embryo development
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TYNYKULOV, M. K., A. B. AKHMETOVA, A. B. ABZHALELOV, Sh E. ARYSTANOVA, A. U. UTAUBAYEVA, Zh N. UALIAKHMETOVA, А. U. TUYAKBAYEVA, and А. Zh ТEMIRKHANOV. "КУЛЬТУРА РАСТИТЕЛЬНЫХ КЛЕТОК IN VITRO AJUGA TURKESTANICA." МИКРОБИОЛОГИЯ ЖӘНЕ ВИРУСОЛОГИЯ, no. 2(45) (June 19, 2024): 159–80. http://dx.doi.org/10.53729/mv-as.2024.02.10.
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The article presents the growth rates of cells of new lines obtained by inoculation-strains of the culture of the endemic plant Ajuga turkestanica. Scientific research was conducted in 2022 in the laboratory of biology of cultured cells of the Institute of Plant Physiology named after K.A. Timiryazev. The purpose of the work: to obtain new tissue and cell culture lines in vitro of the endemic Ajuga turkestanica plant, as well as to study the possibility of regulating culture growth and biosynthesis of secondary metabolism products by the influence of various factors. The objects of the study are suspension cultures of Ajuga turkestanica cells. Source material: callus strain Ajuga turkestanica (Regel) Briq. At21, received in 2016, as well as ready-made frozen collection bases from the cryobank of the Timiryazev IGF of the Russian Academy of Sciences. Research methods: Obtaining a suspension of cells from callus tissue; cultivation of suspension cultures; determination of growth characteristics of the culture: the content of dry and raw biomass in a liter of medium, the concentration of cells in the medium, the viability of the culture. The results of the evaluation of the growth characteristics of suspension crops were obtained. Practical significance: the obtained suspension cultures can be used for various biotechnological studies, such as the production of biologically active substances, the study of secondary metabolism, the development of biotechnological methods for the production of medicines. В статье приведены ростовые показатели клеток новых полученных путем инокуляции линий-штаммов культуры эндемичного растения Ajuga turkestanica. Научные исследования проведены в 2022 году в лаборатории биологии культивируемых клеток Института физиологи растений им. К.А. Тимирязева. Цель работы: получение новых линий культуры тканей и клеток in vitro эндемичного растения Ajuga turkestanica, а также изучение возможности регуляции роста культуры и биосинтеза продуктов вторичного метаболизма воздействием различных факторов. Объектами исследованияявляются суспензионные культуры клеток Ajuga turkestanica. Исходный материал: каллусный штамм Ajuga turkestanica (Regel) Briq. Ат21, полученный в 2016 году, а также готовые замороженные коллекционные базы из криобанка ИФР им. К.А. Тимирязева РАН РФ. Методы исследования: получение суспензии клеток из каллусной ткани; культивирование суспензионных культур; определение ростовых характеристик культуры: содержание сухой и сырой биомассы в литре среды, концентрация клеток в среде, жизнеспособность культуры. Получены результаты по оценке ростовых характеристиках суспензионных культур. Практическая значимость: полученные суспензионные культуры могут быть использованы для различных биотехнологических исследований, таких как получение биологически активных веществ, изучение вторичного метаболизма, разработка биотехнологических методов производства лекарственных препаратов.
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Jarrahy, Reza, Weibiao Huang, George H. Rudkin, Jane M. Lee, Kenji Ishida, Micah D. Berry, Modar Sukkarieh, Benjamin M. Wu, Dean T. Yamaguchi, and Timothy A. Miller. "Osteogenic differentiation is inhibited and angiogenic expression is enhanced in MC3T3-E1 cells cultured on three-dimensional scaffolds." American Journal of Physiology-Cell Physiology 289, no. 2 (August 2005): C408—C414. http://dx.doi.org/10.1152/ajpcell.00196.2004.
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Osteogenic differentiation of osteoprogenitor cells in three-dimensional (3D) in vitro culture remains poorly understood. Using quantitative real-time RT-PCR techniques, we examined mRNA expression of alkaline phosphatase, osteocalcin, and vascular endothelial growth factor (VEGF) in murine preosteoblastic MC3T3-E1 cells cultured for 48 h and 14 days on conventional two-dimensional (2D) poly(l-lactide-co-glycolide) (PLGA) films and 3D PLGA scaffolds. Differences in VEGF secretion and function between 2D and 3D culture systems were examined using Western blots and an in vitro Matrigel-based angiogenesis assay. Expression of both alkaline phosphatase and osteocalcin in cells cultured on 3D scaffolds was significantly downregulated relative to 2D controls in 48 h and 14 day cultures. In contrast, elevated levels of VEGF expression in 3D culture were noted at every time point in short- and long-term culture. VEGF protein secretion in 3D cultures was triple the amount of secretion observed in 2D controls. Conditioned medium from 3D cultures induced an enhanced level of angiogenic activity, as evidenced by increases in branch points observed in in vitro angiogenesis assays. These results collectively indicate that MC3T3-E1 cells commit to osteogenic differentiation at a slower rate when cultured on 3D PLGA scaffolds and that VEGF is preferentially expressed by these cells when they are cultured in three dimensions.
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Colombo, Martina, Isa Mohammed Alkali, Sylwia Prochowska, and Gaia Cecilia Luvoni. "Fighting Like Cats and Dogs: Challenges in Domestic Carnivore Oocyte Development and Promises of Innovative Culture Systems." Animals 11, no. 7 (July 19, 2021): 2135. http://dx.doi.org/10.3390/ani11072135.
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In vitro embryo production in cats and dogs still presents some challenges, and it needs to be optimized to transfer efficient protocols to related wild, endangered species. While the chemical composition of culture media has been the focus of several studies, the importance of culture substrates for oocyte and embryo culture has often been neglected. Traditional in vitro systems, i.e., two-dimensional cultures, do not resemble the physiological environments where cells develop, and they may cause morphological and functional alterations to oocytes and embryos. More modern three-dimensional and microfluidic culture system better mimic the structure and the stimuli found in in vivo conditions, and they could better support the development of oocytes and embryos in vitro, as well as the maintenance of more physiological behaviors. This review describes the different culture systems tested for domestic carnivore reproductive cells along the years, and it summarizes their effects on cultured cells with the purpose of analyzing innovative options to improve in vitro embryo production outcomes.
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Thakur, Anirudh, G. S. Sidhu, and Harminder Singh. "In vitro multiplication of peach rootstocks." Indian Journal of Horticulture 80, no. 03 (September 25, 2023): 251–57. http://dx.doi.org/10.58993/ijh/2023.80.3.4.
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The leaf yellowing and senescence during micro-propagation reduces the shoot proliferation rates with every sub-culture in peach rootstock (Prunus spp.). The effect of culture media and supplements on the proliferation of cultures during the micro-propagation of two peach rootstocks ‘Sharbati’ and ‘Flordaguard’ was studied. In both Prunus genotypes, the proliferated cultures decreased from 77.3% and 67.0 % after the first sub-culture to 35.3 % and 27.3 % after the third sub-culture in ‘Sharbati’ and ‘Flordaguard’, respectively. QL medium significantly improved the proportion of proliferated cultures over MS, WPM, DKW and the modified MS media. The highest proliferated cultures (79.0% and 70.7%) and shoot number per culture (4.2 and 3.7) were recorded after the third subculture, in ‘Sharbati’ and ‘Flordaguard’, respectively with QL medium. Both the rootstocks varied in their response to iron chelates. The higher proliferation rates were obtained with MS medium by substituting Fe-EDTA (0.1 mM) with Fe-EDDHA (0.1-0.3 mM) in ‘Sharbati’ and with Fe-Na EDTA (0.1 mM) in ‘Flordaguard’. In‘Sharbati’, the highest proliferated cultures (44.3%) after the third subculture were observed with Fe-EDDHA (0.3 mM). In ‘Flordaguard’, the highest proportion of proliferated cultures (45%) after the third subculture was recorded with Fe-Na EDTA (0.1 mM). Adding silver nitrate (3 mM) improved the shoot proliferation rates to the highest levels (63% and 51%) after third subculture in ‘Sharbati’ and ‘Flordaguard’, respectively. The highest rooting (34.3% and 38.0%) and number of roots per shoot (2.2 and 2.5) were recorded with 3.75 mM of IBA in ‘Sharbati’ and ‘Flordaguard’, respectively.
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Gupta, P. S. P., H. S. Ramesh, B. M. Manjunatha, S. Nandi, and J. P. Ravindra. "Production of buffalo embryos using oocytes from in vitro grown preantral follicles." Zygote 16, no. 1 (February 2008): 57–63. http://dx.doi.org/10.1017/s096719940700442x.
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SummaryThe present study examines the use of buffalo preantral follicles as a source of oocytes for in vitro embryo production. Preantral follicles were isolated from abattoir-derived buffalo ovaries and were grown for 100 days in five different culture systems: (1) minimum essential medium (MEM); (2) coconut water; (3) MEM + ovarian mesenchymal cell (OMC) co-culture; (4) MEM + granulosa cell (GC) co-culture; or (5) MEM + cumulus cell (CC) co-culture. Low growth rates for the preantral follicles were observed when follicles were cultured in MEM or coconut water medium. Moderate growth rates were seen for OMC and GC co-cultures, and high rates of growth were observed when follicles were grown in CC co-culture. The survival of preantral follicles was low in the MEM culture (<25%), but was over 75% in the other culture systems. Oocytes were not recovered from the MEM group, while an oocyte recovery rate of 80–100% was observed when the follicles were cultured with coconut water/somatic cells. Transferable embryos could be produced only with the oocytes obtained from preantral follicles grown in the OMC and CC co-culture systems. This study demonstrates, for the first time, that it is possible to produce buffalo embryos by in vitro fertilization of oocytes derived from in vitro grown preantral follicles.
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Haselager, Marco, Eduard Perelaer, Arnon P. Kater, and Eric Eldering. "Development of a Novel Lymph Node-Based 3D Culture System Promoting Chronic Lymphocytic Leukemia Proliferation and Survival." Blood 136, Supplement 1 (November 5, 2020): 47–48. http://dx.doi.org/10.1182/blood-2020-141962.
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INTRODUCTION. Primary chronic lymphocytic leukemia (CLL) cells, despite originating from a proliferative disease, rapidly undergo apoptosis in vitro in absence of microenvironmental survival signals1. Although co-culture with stromal cells or the addition of soluble factors can increase and extend CLL survival, no system permits the long-term expansion of CLL cells in vitro2. The difficulties of mimicking a physiologic microenvironment supporting CLL cells hinder in vitro studies of proliferation, drug screens and prevent propagation of rare subclones. For other cancers, various types of 3D cultures have been introduced utilizing scaffolds, gels, spheroid cultures and fluidic systems, representing a more accurate representation of the in vivo microenvironment3. Unlike solid tumors, secondary lymphoid tissues where CLL cells proliferate in vivo, do not derive from a single stem cell progenitor. Developing an appropriate 3D in vitro culture system for CLL is of obvious importance and may contribute pathophysiological relevance to study long-term CLL proliferation and more accurate drug screening4,5. Within the field of CLL, attempts have focused on bone marrow stroma, but it may be biologically and clinically more relevant to investigate the lymph node niche as this is the critical site of CLL proliferation6. METHODS. Primary CLL cells were cultured in various 3D systems including hydrogels, hanging drop cultures and ultra-low attachment plates (ULA) plates in parallel to an optimal 2D system, consisting of the culture of primary CLL cells on a monolayer of CD40L-presenting fibroblasts (3T40) or 3T3 negative control fibroblasts. CLL cells were either cultured as PBMCs alone, with or without T cells, or co-cultured with 3T40 or primary lymph node fibroblasts. CLL cells were either stimulated directly with IL-2, IL-15, IL-21 and CpG and/or indirectly via a T cell stimulation of anti-CD3/CD28. RESULTS. After testing and comparing multiple systems for the in vitro culture of CLL cells, we optimized a novel CLL culture system utilizing ULA plates creating spheroids of PBMCs isolated from peripheral blood. Without the addition of soluble factors or stroma, primary CLL cells in the ULA 3D model could be maintained in culture for 6 weeks as opposed to 1 week in the 2D system. Aside from significantly promoting CLL survival, cultures could be expanded approximately 3-4-fold over a course of 6 weeks using the ULA 3D model. 3D cultures showed a more consistent and significantly increased CLL proliferation compared to 2D cultures, independent of IGHV mutation status, increasing the average proliferation index of 2.87 to 3.90 (n=10). Additionally, co-culture with LN-derived stromal cells further increased CLL proliferation, reaching a maximum of 8 generations (n=6) (Figure 1). Lastly, when PBMCs were stimulated with IL-2, IL-15, IL-21 and CpG, spheroids developed proliferation center-like structures after 4 weeks of culture. CONCLUSIONS. We established a lymph node-based 3D in vitro culture system for CLL leading to increased CLL proliferation and survival compared to 2D systems. The set-up allows long-term expansion of CLL cells in vitro, as well as formation of proliferation center-like structures. We are currently optimizing drug resistance studies, expansion of specific CLL subclones and performing competition experiments. References: 1. Hamilton et al., Mimicking the tumour microenvironment: three different co-culture systems induce a similar phenotype but distinct proliferative signals in primary chronic lymphocytic leukaemia cells, 2012. 2. Asslaber et al., Mimicking the microenvironment in chronic lymphocytic leukaemia - where does the journey go?, 2013. 3. Gurski et al., 3D Matrices for Anti-Cancer Drug Testing and Development, 2010. 4. Nunes et al., 3D tumor spheroids as in vitro models to mimic in vivo human solid tumors resistance to therapeutic drugs, 2019. 5. Aljitwai et al., A novel three-dimensional stromal-based model for in vitro chemotherapy sensitivity testing of leukemia cells, 2014. 6. Van Gent et al., In vivo dynamics of stable chronic lymphocytic leukemia inversely correlates with somatic hypermutation levels and suggest no major leukemic turnover in bone marrow, 2008. Disclosures Kater: Genentech: Research Funding; Abbvie: Research Funding; Roche: Research Funding; Janssen: Research Funding; Celgene: Research Funding. Eldering:Celgene: Research Funding; Janssen: Research Funding; Genentech: Research Funding.
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Adelberg, J., M. Kroggel, and J. Toler. "Physical Environment in vitro Affects Laboratory and Nursery Growth of Micropropagated Hostas." HortTechnology 10, no. 4 (January 2000): 754–57. http://dx.doi.org/10.21273/horttech.10.4.754.
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Hosta ×hybrid Tratt. `Blue Cadet' and Hosta tokudama Tratt. `Newberry Gold' were micropropagated in shaken liquid culture and on agar media, in conventional vessels and vessels modified for ventilation in vitro. Acclimatization under intermittent mist and growth in an outdoor nursery during the late spring and summer were monitored by dry weight analysis of sample plants every 4 days for a 60-day period (ex vitro growth). Results for `Newberry Gold' were 1) in vitro shoot growth was greater in liquid than agar culture, regardless of vessel; 2) shoots from agar or liquid culture grew at similar rates ex vitro; 3) ex vitro root growth was greater for liquid than agar cultured plants, regardless of vessel type. Results for `Blue Cadet' were 1) in vitro and ex vitro shoot growth was greater in liquid than agar culture regardless of vessel type and 2) ex vitro root growth was greatest for liquid cultured plants from conventional vessels. Ventilated vessels were generally beneficial for agar but not liquid culture. Benefits of liquid culture for micropropagation of Hosta found in vitro are at least maintained and sometimes enhanced during ex vitro growth in the mist bed and nursery.
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du Preez, Francois, Antoinette Paula Malan, and Pia Addison. "Potential of in vivo- and in vitro-cultured entomopathogenic nematodes to infect Lobesia vanillana (Lepidoptera: Tortricidae) under laboratory conditions." PLOS ONE 16, no. 8 (August 16, 2021): e0242645. http://dx.doi.org/10.1371/journal.pone.0242645.
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Entomopathogenic nematodes (EPNs) have been successfully applied as biological control agents against above ground and soil stages of insect pests. However, for commercial application, it is crucial to mass culture these nematodes using in vitro liquid culture technology, as it is not attainable when using susceptible insects as hosts. Lobesia vanillana (Lepidoptera: Tortricidae) is regarded a sporadic pest of wine grapes in South Africa. The in vivo- and in vitro-cultured South African EPNs, Steinernema yirgalemense and Steinernema jeffreyense (Rhabditida: Steinernematidae), were evaluated against larvae and pupae of L. vanillana in laboratory bioassays. For larvae, high mortality was observed for all treatments: In vitro-cultured S. yirgalemense (98%) performed better than S. jeffreyense (73%), while within in vivo cultures, there was no difference between nematode species (both 83%). No significant difference was detected between in vivo- and in vitro cultures of the same nematode species. The LD50 of the in vitro-cultured S. yirgalemense, was 7.33 nematodes per larva. Mortality by infection was established by dissecting L. vanillana cadavers and confirming the presence of nematodes, which was > 90% for all treatments. Within in vitro cultures, both S. yirgalemense and S. jeffreyense were able to produce a new cohort of infective juveniles from L. vanillana larvae. Pupae, however, were found to be considerably less susceptible to EPN infection. This is the first study on the use of EPNs to control L. vanillana. The relative success of in vitro-cultured EPN species in laboratory assays, without any loss in pathogenicity, is encouraging for further research and development of this technology.
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Dewir, Yaser Hassan, Abdulla Alsadon, Ahmed Ali Al-Aizari, and Mohaidib Al-Mohidib. "In Vitro Floral Emergence and Improved Formation of Saffron Daughter Corms." Horticulturae 8, no. 10 (October 20, 2022): 973. http://dx.doi.org/10.3390/horticulturae8100973.
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In vitro cormogenesis is a potential tool for improving saffron production under controlled conditions. In this study, the effects of explant type, culture type, and medium supplements on saffron daughter corm formation in vitro were assessed. Saffron flowers emerged 30 days after culture, and the sizes of in-vitro- and ex-vitro-produced flowers and stigmas were similar. In vitro daughter corm formation and the saffron life cycle was completed after 10 and 14 weeks of culture, respectively. Using in vitro intact corms was more effective for corm production than using apical buds. Compared with apical bud explants, mother corm explants produced more corms with a higher fresh weight and diameter. Compared with solid culture, liquid cultures using bioreactors provided corms with a higher fresh weight and diameter, regardless of explant type. An ebb and flow system provided the highest cormlet fresh weight and diameter but the fewest cormlets, whereas an immersion system provided more cormlets with a smaller size. Saffron apical buds cultured with salicylic acid at 75 mg L−1 or glutamine at 600 mg L−1 exhibited the highest cormlet diameter and fresh weight. These findings will improve the process of in vitro cormogenesis and the production of saffron under controlled conditions.
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Romberger, D. J., P. Pladsen, L. Claassen, M. Yoshida, J. D. Beckmann, and S. I. Rennard. "Insulin modulation of bronchial epithelial cell fibronectin in vitro." American Journal of Physiology-Lung Cellular and Molecular Physiology 268, no. 2 (February 1, 1995): L230—L238. http://dx.doi.org/10.1152/ajplung.1995.268.2.l230.
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Fibronectin (Fn) is involved in the migration of epithelial cells in re-epithelialization of wounds. Epithelial cell-derived Fn is particularly potent as a chemotactic factor for bronchial epithelial cells (BECs) in vitro. Thus modulation of airway epithelial cell Fn may be a key aspect of airway repair. Insulin is both an important growth factor and known chemotactic factor for cultured BECs. We postulated that insulin may modulate Fn production of cultured BECs. We examined this hypothesis utilizing bovine BECs in culture with serum-free media with and without insulin. BECs grown in media without insulin released more Fn into culture supernatants and contained more Fn in cell layers than cells grown with insulin. Labeling of cells with [35S]methionine demonstrated an increase in new protein production and Fn mRNA expression was increased. Increased Fn in BEC cultures without insulin was associated with an increase in active transforming growth factor-beta (TGF-beta) release as measured by a standard bioassay. Increased BEC Fn in cultures without insulin was partially inhibited by exposure of cultures to TGF-beta antibody. Thus insulin appears to modulate BEC Fn production in vitro in part through a TGF-beta-dependent mechanism. Insulin may be involved in airway repair mechanisms through modulation of epithelial cell Fn production.
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Zanon, Renata, Amauri Pierucci, and Alexandre Oliveira. "Interferon beta and glatiramer acetate induce proliferation of Schwann cells in vitro." Acta Neurobiologiae Experimentalis 69, no. 1 (March 31, 2009): 146–52. http://dx.doi.org/10.55782/ane-2009-1737.
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Techniques as well as substances capable of stimulating cultured Schwann cell (SC) proliferation are needed for future therapeutical applications. In this work, the effects of interferon beta (IFNbeta) and glatiramer acetate (GA) on SC cultures were tested, with interest on the growth curve and potential proliferative effects. Primary cultures were prepared from the sciatic nerves of neonatal rats and seeded onto culture plates. Such cells were then subjected to treatment with different doses of IFN beta (100, 500 and 1000 IU/ml) and of GA (1.2, 2.5 and 5.0 mmg/ml) for five consecutive days. S100beta and DAPI double labeling was used in order to confirm the purity of the cultures. Both treatment with IFN and GA resulted in an increased number of cultured SCs. However, only IFN beta induced a statistically significant proliferative outcome. Such results indicate that addition of IFN beta to the culture medium is efficient in order to improve SC proliferation in vitro.
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Sansberro, Pedro A., Hebe Y. Rey, and Luis A. Mroginski. "In Vitro Culture of Zygotic Embryos of Ilex Species." HortScience 36, no. 2 (April 2001): 351–52. http://dx.doi.org/10.21273/hortsci.36.2.351.
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Plants of Ilex argentina L., I. brasiliensis (S.) L., I. brevicuspis R., I. dumosa R., I. integerrima (V.C.) L., I. microdonta R., I. pseudoboxus R., and I. theezans C.M. were obtained by immature embryo culture. Heart-stage zygotic embryos were removed from immature fruits and cultured aseptically on quarter-strength Murashige and Skoog medium with 3% sucrose, 0.65% agar, and 0.1 mg·L-1 zeatin. Cultures were incubated at 27±2°C for 4 weeks, in darkness and subsequently transferred to a culture room with a 14-hour photoperiod (116 μmol·m-2·s-1) for another 4 weeks. Seedlings with two leaves, derived from germinated embryos, were successfully transplanted to pots containing 1 peat: 1 perlite: 1 sand (v/v) and were maintained in greenhouse conditions. From 95% to 100% of transplanted seedlings survived. Chemical name used: 6-(4-hydroxy-3-methylbut-2-enylamino) purine (zeatin).
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Al-Juboory, Karim H., and Jabar H. Al-Niami. "Adventitious Shoot and Plantlet Formation from Cultured Apple Leaf Explants." HortScience 31, no. 4 (August 1996): 629f—629. http://dx.doi.org/10.21273/hortsci.31.4.629f.
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Leaves of wild apple (Malus domestica Borkh) were excised from in vitro grown shoots transversely cut into halves and plated onto petri dishes containing regeneration media. Cultures were kept in the dark for three weeks before adventitious shoots were observed. Callus from leaf explants produced adventitious shoots after 3 months of in vitro culture. Callus were cultured on Nitsch and Nitsch medium supplemented with a range of BA (0.0–2.0 μm) and NAA (0.0–10 μm). BA at 10 μm combined with NAA (0.5 μm) proved most effective for stimulating shoot proliferation of cultured apple. Plantlets from tissue culture were easily transferred to the greenhouse environment.
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Racusen, L. C., B. A. Fivush, H. Andersson, and W. A. Gahl. "Culture of renal tubular cells from the urine of patients with nephropathic cystinosis." Journal of the American Society of Nephrology 1, no. 8 (February 1991): 1028–33. http://dx.doi.org/10.1681/asn.v181028.
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Nephropathic cystinosis represents a prototype for lysosomal storage diseases and is the most common cause of renal tubular Fanconi's syndrome. Mechanisms of the tubular transport defects in this disease have not been defined, however, in part because the cells readily cultured from affected patients, leukocytes and fibroblasts, do not express epithelial transport functions. Except for a single autopsy report, renal tubular cells from these patients have not been studied in vitro. In these studies, noninvasive harvesting and culture of renal tubular cells from the urine of patients with cystinosis is described. Cultures of renal tubular cells could be established from over 50% of the isolates which contained viable cells and which remained uncontaminated in vitro. Cells had an epithelial morphology in culture, and the majority of cultured cells expressed proximal tubular brush border marker enzyme. Cultured cells also expressed the storage defect in vitro, containing cystine levels up to 100 times those of normal cells. Cultured cells could be depleted of cystine by using the thiol cysteamine. This in vitro model system should be very useful for studying the mechanisms of renal tubular transport defects in this disease.
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Miyamoto, Yoshitaka, Masashi Ikeuchi, Hirofumi Noguchi, Tohru Yagi, and Shuji Hayashi. "Enhanced Adipogenic Differentiation of Human Adipose-Derived Stem Cells in an in vitro Microenvironment: The Preparation of Adipose-Like Microtissues Using a Three-Dimensional Culture." Cell Medicine 9, no. 1-2 (January 2017): 35–44. http://dx.doi.org/10.3727/215517916x693096.
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The application of stem cells for cell therapy has been extensively studied in recent years. Among the various types of stem cells, human adipose tissue-derived stem cells (ASCs) can be obtained in large quantities with relatively few passages, and they possess a stable quality. ASCs can differentiate into a number of cell types, such as adipose cells and ectodermal cells. We therefore focused on the in vitro microenvironment required for such differentiation and attempted to induce the differentiation of human stem cells into microtissues using a microelectromechanical system. We first evaluated the adipogenic differentiation of human ASC spheroids in a three-dimensional (3D) culture. We then created the in vitro microenvironment using a 3D combinatorial TASCL device and attempted to induce the adipogenic differentiation of human ASCs. The differentiation of human ASC spheroids cultured in maintenance medium and those cultured in adipocyte differentiation medium was evaluated via Oil red O staining using lipid droplets based on the quantity of accumulated triglycerides. The differentiation was confirmed in both media, but the human ASCs in the 3D cultures contained higher amounts of triglycerides than those in the 2D cultures. In the short culture period, greater adipogenic differentiation was observed in the 3D cultures than in the 2D cultures. The 3D culture using the TASCL device with adipogenic differentiation medium promoted greater differentiation of human ASCs into adipogenic lineages than either a 2D culture or a culture using a maintenance medium. In summary, the TASCL device created a hospitable in vitro microenvironment and may therefore be a useful tool for the induction of differentiation in 3D culture. The resultant human ASC spheroids were “adipose-like microtissues” that formed spherical aggregation perfectly and are expected to be applicable in regenerative medicine as well as cell transplantation.
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Stimart, Dennis P., John C. Mather, and Kenneth R. Schroeder. "Shoot Proliferation and Rooting in Vitro of Pulmonaria." HortScience 33, no. 2 (April 1998): 339–41. http://dx.doi.org/10.21273/hortsci.33.2.339.
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Expanding shoot tips of Pulmonaria `Roy Davidson' and Pulmonaria saccharata `Margery Fish' were cultured in vitro on a modified Murashige and Skoog medium containing BA to establish proliferating cultures for use in comparing BA concentrations on shoot proliferation and rooting. The optimum level for shoot proliferation was 8.8 μm BA. Greatest rooting was on medium without BA. Genotype and time in culture influenced shoot and root counts. Chemical names used: N6-benzyladenine (BA)
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Stimart, Dennis P., John C. Mather, and Kenneth R. Schroeder. "Shoot Proliferation and Rooting in Vitro of Pulmonaria." HortScience 33, no. 2 (April 1998): 339–41. http://dx.doi.org/10.21273/hortsci.33.2.0339.
Full textAbstract:
Expanding shoot tips of Pulmonaria `Roy Davidson' and Pulmonaria saccharata `Margery Fish' were cultured in vitro on a modified Murashige and Skoog medium containing BA to establish proliferating cultures for use in comparing BA concentrations on shoot proliferation and rooting. The optimum level for shoot proliferation was 8.8 μm BA. Greatest rooting was on medium without BA. Genotype and time in culture influenced shoot and root counts. Chemical names used: N6-benzyladenine (BA)
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Hettiarachchi, H. D. B. K., V. R. M. Vidhanaarachchi, S. P. N. C. Jayarathna, and Perera Dinum. "Effect of exogenous polyamines on coconut (<em>Cocos nucifera</em> L.) embryogenic callus multiplication." COCOS 23, no. 1 (December 30, 2022): 47–56. http://dx.doi.org/10.4038/cocos.v23i1.5823.
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Major bottlenecks of coconut in vitro culture are poor plant regeneration rate, severe browning and premature necrosis of cultured tissue, and heterogeneous response of individual palms and explants to in vitro culture conditions. Among them, tissue browning is a common and often severe problem in coconut in vitro culture systems which results in death of explant/ callus ultimately. This experiment was carried out to enhance the in vitro multiplication of coconut, which is a highly recalcitrant species to in vitro culture through exogenously added polyamines in the media. The polyamines are important for in vitro cell division, cell growth and to delay senescence. In the present study, unfertilized ovary derived calli were cultured on Y3 basal medium supplemented with sucrose (5%), 2,4-D, phytagel (3 gL-1), activated charcoal (2.5 gL-1), and polyamine. Three polyamine types (O.lmM spermine, 1.0 mM putrescine and 0.5mM spermidine) were tested in combination with two 2,4-D concentrations (0.30 and 0.60 mM) in order to enhance coconut in vitro multiplication. All the cultures were incubated in dark at 26±2 °C. The embryogenic structures, embryogenic callusing, non-embryogenic callusing, and browning were recorded separately for each treatment after five weeks from the culture establishment. The polyamine treatments did not have significant effects on frequencies of embryogenic structures, embryogenic callus, non-embryogenic callus and browned callus formation at the initial stages of coconut somatic embryogenesis irrespective of the tested 2,4-D concentrations. Furthermore, the results indicated that decreased 2,4-D levels have significantly reduced browning, resulting 44.79 % browning frequency (0.8-fold lesser browning) in media supplemented with 0.30 mM 2,4-D. However, the potential effects of exogenously added polyamines at the initial stages of coconut somatic embryogenesis could be delivered during the latter stages of the somatic embryo genesis as previously reported in other experiments. Thus, continuous subculture may be necessary.
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Orihuela, Carlos J., Rob Janssen, Christopher W. Robb, David A. Watson, and David W. Niesel. "Peritoneal Culture Alters Streptococcus pneumoniae Protein Profiles and Virulence Properties." Infection and Immunity 68, no. 10 (October 1, 2000): 6082–86. http://dx.doi.org/10.1128/iai.68.10.6082-6086.2000.
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ABSTRACT We have examined the properties of Streptococcus pneumoniae cultured in the murine peritoneal cavity and compared its virulence-associated characteristics to those of cultures grown in vitro. Analysis of mRNA levels for specific virulence factors demonstrated a 2.8-fold increase in ply expression and a 2.2-fold increase in capA3 expression during murine peritoneal culture (MPC). Two-dimensional gels and immunoblots using convalescent-phase patient sera and murine sera revealed distinct differences in protein production in vivo (MPC). MPC-grown pneumococci adhered to A549 epithelial cell lines at levels 10-fold greater than those cultured in vitro.
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Marrero-Berrios, Ileana, Anil Shrirao, Charles P. Rabolli, Rishabh Hirday, Rene S. Schloss, and Martin L. Yarmush. "Multi-layer stackable tissue culture platform for 3D co-culture." TECHNOLOGY 08, no. 01n02 (March 2020): 37–49. http://dx.doi.org/10.1142/s233954782050003x.
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In vitro tools, which can enable development of models that replicate the cell microenvironment associated with complex diseases such as osteoarthritis (OA), are critically needed. In OA, catabolic and inflammatory processes orchestrated by multiple cell types lead to the eventual destruction of articular cartilage. To address this need, our group developed a device that will enable investigation of complex cell systems. Our stackable tissue culture insert was fabricated and characterized with respect to biocompatibility, ease of use, and potential for tissue culture applications. The stackable tissue culture inserts can be easily modified, fabricated, and assembled into commercially available multi-well plates. In vitro studies conducted with three different cell types demonstrated high cell viability and functional secretion when cultured in the stackable inserts. Furthermore, synergistic effects when the three cell types were cultured together were observed. This demonstrates the need to more fully interrogate in vitro culture systems, and this stackable insert can provide a tool to fill the current technological void to do so.
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Huttala, Outi, Desiree Loreth, Synnöve Staff, Minna Tanner, Harriet Wikman, and Timo Ylikomi. "Decellularized In Vitro Capillaries for Studies of Metastatic Tendency and Selection of Treatment." Biomedicines 10, no. 2 (January 26, 2022): 271. http://dx.doi.org/10.3390/biomedicines10020271.
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Vascularization plays an important role in the microenvironment of the tumor. Therefore, it should be a key element to be considered in the development of in vitro cancer assays. In this study, we decellularized in vitro capillaries to remove genetic material and optimized the medium used to increase the robustness and versatility of applications. The growth pattern and drug responses of cancer cell lines and patient-derived primary cells were studied on decellularized capillaries. Interestingly, two distinct growth patterns were seen when cancer cells were grown on decellularized capillaries: “network” and “cluster”. Network formation correlated with the metastatic properties of the cells and cluster formation was observed in non-metastatic cells. Drug responses of patient-derived cells correlated better with clinical findings when cells were cultured on decellularized capillaries compared with those cultured on plastic. Decellularized capillaries provide a novel method for cancer cell culture applications. It bridges the gap between complex 3D culture methods and traditional 2D culture methods by providing the ease and robustness of 2D culture as well as an in vivo-like microenvironment and scaffolding for 3D cultures.
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Kovalenko, O. Hr, A. M. Kyrychenko, and O. Yu Kovalenko. "Virus Infected Bean Tissue Culture Cells and It’s Healing in vitro Using Liposomal form of Glycanes." Mikrobiolohichnyi Zhurnal 82, no. 5 (October 17, 2020): 58–64. http://dx.doi.org/10.15407/microbiolj82.05.058.
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The aim of the study was to develop a recovery means for beans infected by Bean yellow mosaic virus (BYMV) as well as Bean common mosaic virus (BCMV) using callus culture and liposomal glycan preparations. Methods. Cultivation of explants and callus cultures was carried out in vitro using conventional methods of plant biotechnology. The tissue culture propagation was performed during the spring or summer seasons. The presence of viral infection was tested by reverse transcription polymerase chain reaction. The virus-specific primers that allowed amplifying the conserved regions of the capsid protein gene of BCMV or BYMV were used for virus identification. Results. The culture of bean callus infected with BCMV was obtained and adapted for antiviral agents testing. It has been shown that during long-term cultivation (10–12 weeks) in the presence of liposomal preparation containing Ganoderma adspersum glucan (10–100 mg/l), plant tissue culture become free from viruses following virus eradication. This is evidenced by the absence in the callus tissue of 391 bp sequences typical for the virus coat protein gene. Conclusions. The full suppression of virus reproduction and gradual elimination of virus occurred in callus tissue obtained from BCMV-infected beans and cultured on B-5 medium supplemented with liposomal glycanglycolipid complex (10–100 mg/l). The data obtained can be useful for the development of practical control method to cure plant virus diseases using callus culture and antiviral-active glycan-glycolipid complexes.
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Carrara, Maria, Lorenzo Cima, Roberto Cerini, and Maurizio Dalle Carbonare. "A New In Vitro Model for Predicting the Toxicity of Cosmetic Products." Alternatives to Laboratory Animals 20, no. 1 (January 1992): 138–43. http://dx.doi.org/10.1177/026119299202000119.
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A method has been developed whereby cosmetic products which are not soluble in water or in alcohol can be brought into contact with cell cultures by being placed in a cell culture insert, which is then placed in the cell culture well. Preliminary experiments were carried out with L929 cells, and cytotoxicity was evaluated by measuring neutral red uptake and the total protein content of treated cultured cells. Encouraging results were obtained in comparisons of three cosmetic emulsions and of one emulsion containing a range of concentrations of two preservatives, Kathon CG and Bronopol.
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Anderson, A. R., D. Taylor, E. A. Williams, and F. Arredondo. "Simplified culture conditions: comparing invocell culture device to in vitro culture." Fertility and Sterility 108, no. 3 (September 2017): e110. http://dx.doi.org/10.1016/j.fertnstert.2017.07.336.
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Smith-Thomas, L. C., and J. W. Fawcett. "Expression of Schwann cell markers by mammalian neural crest cells in vitro." Development 105, no. 2 (February 1, 1989): 251–62. http://dx.doi.org/10.1242/dev.105.2.251.
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During embryonic development, neural crest cells differentiate into a wide variety of cell types including Schwann cells of the peripheral nervous system. In order to establish when neural crest cells first start to express a Schwann cell phenotype immunocytochemical techniques were used to examine rat premigratory neural crest cell cultures for the presence of Schwann cell markers. Cultures were fixed for immunocytochemistry after culture periods ranging from 1 to 24 days. Neural crest cells were identified by their morphology and any neural tube cells remaining in the cultures were identified by their epithelial morphology and immunocytochemically. As early as 1 to 2 days in culture, approximately one third of the neural crest cells stained with m217c, a monoclonal antibody that appears to recognize the same antigen as rat neural antigen-1 (RAN-1). A similar proportion of cells were immunoreactive in cultures stained with 192-IgG, a monoclonal antibody that recognizes the rat nerve growth factor receptor. The number of immunoreactive cells increased with time in culture. After 16 days in culture, nests of cells, many of which had a bipolar morphology, were present in the area previously occupied by neural crest cells. The cells in the nests were often associated with neurons and were immunoreactive for m217c, 192-IgG and antibody to S-100 protein and laminin, indicating that the cells were Schwann cells. At all culture periods examined, neural crest cells did not express glial fibrillary acidic protein. These results demonstrate that cultured premigratory neural crest cells express early Schwann cell markers and that some of these cells differentiate into Schwann cells. These observations suggest that some neural crest cells in vivo may be committed to forming Schwann cells and will do so provided that they then proceed to encounter the correct environmental cues during embryonic development.
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Mercer, J. G., A. E. Munn, J. W. Smith, and H. H. Rees. "Cuticle production and ecdysis in larval marine ascaridoid nematodes in vitro." Parasitology 92, no. 3 (June 1986): 711–20. http://dx.doi.org/10.1017/s0031182000065562.
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SUMMARYInfective 3rd-stage larvae of three species of marine ascaridoid nematodes, Pseudoterranova (= Phocanema = Porrocaecum = Terranova) decipiens, Anisakis simplex and Phocascaris/Contracaecum sp. were cultured in vitro in 0·9% saline or Medium 199 at 37°C beneath an atmosphere of either air or 95% air:5% CO2 All three species responded to culture at 37°C by producing a new cuticle. The majority of P. decipiens completed ecdysis under all the culture conditions employed. Ecdysis in A. simplex was stimulated by a high concentration of CO2; this effect was particularly noticeable in saline cultures. Both A. simplex and Phocascaris/Contracaecum sp. developed more effectively in a nutrient medium than in saline.
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Camper, N. D., P. S. Coker, D. E. Wedge, and R. J. Keese. "In vitro culture of Ginkgo." In Vitro Cellular & Developmental Biology - Plant 33, no. 2 (April 1997): 125–27. http://dx.doi.org/10.1007/s11627-997-0009-7.
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Schinor, Evandro Henrique, Fernando Alves de Azevedo, Francisco de Assis Alves Mourão Filho, and Beatriz Madalena Januzzi Mendes. "In vitro organogenesis in some citrus species." Revista Brasileira de Fruticultura 33, no. 2 (May 27, 2011): 526–31. http://dx.doi.org/10.1590/s0100-29452011005000050.
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In vitro organogenesis of Citrus was studied for the genotypes Citrus sinensis cv. 'Natal', C. limonia, C. volkameriana, and C. aurantium, with the use of epicotyl segments-derived explants, cultured in MT salts and vitamins medium supplemented with different concentrations of 6-benzylaminopurine (BAP - 0.0; 0.5; 1.0; 1.5 or 2.0 mg L-1). For the recalcitrant genotypes C. limonia and C. aurantium the in vitro organogenesis was also studied with internodal segments-derived explants, cultured in MT salts and vitamins medium supplemented with 0; 0.5; 1.0; 2.0, or 4.0 mg L-1 of BAP. The efficiency of culture medium supplementation with the combination of BAP (0.0; 1.0, or 2.0 mg L-1) and NAA (1-naphthaleneacetic acid - 0.0; 0.3, or 0.5 mg L-1) in the development of adventitious shoots was evaluated for C. aurantium. Culture medium supplementation with BAP is not essential for the adventitious shoots development in the four genotypes studied when epicotyl segments-derived explants are used. In general, culture media supplementation with BAP decreased the percentage of responsive explants excepted for C. sinensis cv. 'Natal' and C. limonia when the concentrations of 1.5 and 2.0 mg/L were used. The presence of cytokinin, in concentrations up to 2 mg/L, stimulated the in vitro organogenesis when internodal segments-derived explants were used for C. limonia and C. aurantium. For C. aurantium no adventitious shoots developed in explants (internodal segments) cultured in basal culture medium, without BAP supplementation. Although no statistic differences could be detected, culture media supplementation with the combination of BAP and NAA favored the development of adventitious shoots in C. aurantium. The best concentration of NAA varied according to BAP concentration. The results presented herein, show that Citrus in vitro organogenesis depends on the interaction of culture medium composition, explant differentiation level, and genotype.
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Silva, J. R. V., T. Tharasanit, M. A. M. Taverne, G. C. van der Weijden, R. R. Santos, J. R. Figueiredo, and R. van den Hurk. "The activin-follistatin system and in vitro early follicle development in goats." Journal of Endocrinology 189, no. 1 (April 2006): 113–25. http://dx.doi.org/10.1677/joe.1.06487.
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The aim of the present study was to investigate the effects of activin-A and follistatin on in vitro primordial and primary follicle development in goats. To study primordial follicle development (experiment 1), pieces of ovarian cortex were cultured in vitro for 5 days in minimal essential medium (MEM) supplemented with activin-A (0, 10 or 100 ng/ml), follistatin (0, 10 or 100 ng/ml) or combinations of the two. After culture, the numbers of primordial follicles and more advanced follicle stages were calculated and compared with those in non-cultured tissue. Protein and mRNA expression of activin-A, follistatin, Kit ligand (KL), growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) in non-cultured and cultured follicles were studied by immunohistochemistry and PCR. To evaluate primary follicle growth (experiment 2), freshly isolated follicles were cultured for 6 days in MEM plus 100 ng/ml activin-A, 100 ng/ml follistatin or 100 ng/ml activin-A plus 200 ng/ml follistatin. Morphology, follicle and oocyte diameters in cultured tissue and isolated follicles before and after culture were assessed. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) reactions were performed to study DNA fragmentation in follicles. In experiment 1, it was found that goat primordial follicles were activated to develop into more advanced stages, i.e. intermediate and primary follicles, during in vitro culture, but neither activin-A nor follistatin affected the number of primordial follicles that entered the growth phase. Activin-A treatment enhanced the number of morphologically normal follicles and stimulated their growth during cortical tissue culture. The effects were, however, not counteracted by follistatin. The follicles in cultured goat tissue maintained their expression of proteins and mRNA for activin-A, follistatin, KL, GDF-9 and BMP-15. Fewer than 30% of the atretic follicles in cultured cortical tissue had TUNEL-positive (oocyte or granulosa) cells. Activin-A did not affect the occurrence of TUNEL-positive cells in follicles within cortical tissue. In experiment 2, addition of activin-A to cultured isolated primary follicles significantly stimulated their growth, the effect being counteracted by follistatin. Absence of such a neutralizing effect of follistatin in the cultures with ovarian cortical tissue can be due to lower dose of follistatin used and incomplete blockage of activin in these experiments. In contrast to cortical enclosed atretic follicles, all atretic follicles that had arisen in cultures with isolated primary follicles had TUNEL-positive cells, which points to differences between isolated and ovarian tissue-enclosed follicles with regard to the followed pathways leading to their degeneration. In summary, this in vitro study has demonstrated that cultured goat primordial follicles are activated to grow and develop into intermediate and primary follicles. During in vitro culture, the follicles maintain their ability to express activin-A, follistatin, KL, GDF-9 and BMP-15. The in vitro growth and survival of activated follicles enclosed in cortical tissue and the in vitro growth of isolated primary follicles are stimulated by activin-A.
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Goncharuk, E. A., O. L. Saibel, G. P. Zaitsev, and N. V. Zagoskina. "The Elicitor Effect of Yeast Extract on the Accumulation of Phenolic Compounds in Linum grandiflorum Cells Cultured In Vitro and Their Antiradical Activity." Biology Bulletin 49, no. 6 (December 2022): 620–28. http://dx.doi.org/10.1134/s1062359022060061.
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Abstract This paper examines the elicitor effect of yeast extract (YE) in various concentrations (200–1000 mg/L) on the accumulation of phenolic compounds (PC) in flowering flax (Linum grandiflorum Desf.) cells cultured in vitro and their antiradical activity. It is established that the total PС content and the content of phenylpropanoids increase in the cell culture, especially at high YE concentrations in the medium (500 and 1000 mg/L). The antiradical activity of flax culture extracts remains in most cases at the control level. Therefore, the elicitation of flowering flax in vitro cultures by YE activates the PC biosynthesis resulting in the accumulation of these secondary metabolites, while the antiradical activity of cell culture extracts does not decrease compared to the control level.
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Guo, W., K. Kamiya, J. Cheng, and J. Toyama. "Changes in action potentials and ion currents in long-term cultured neonatal rat ventricular cells." American Journal of Physiology-Cell Physiology 271, no. 1 (July 1, 1996): C93—C102. http://dx.doi.org/10.1152/ajpcell.1996.271.1.c93.
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A primary culture of neonatal ventricular myocytes isolated from day-old rats was established for investigating the changes in action potentials and ion currents over long periods. Cells at days 5 and 15 in culture were studied. These changes in vitro were compared with those in situ derived from the age-matched freshly isolated cells. During primary culture, quiescent cells demonstrated shortening of action potential durations (APD) resembling the developmental changes observed in situ. The beating cultured cells were not associated with APD shortening. Despite constant current amplitudes, the densities of Ca2+ currents (ICa) decreased in the quiescent cultures at later ages as a result of cell enlargement. ICa densities were maintained in the beating cultured and freshly isolated cells. Acceleration in the inactivation of ICa was observed during developments both in vitro and in situ. In addition, the densities of transient outward currents (Ito) tripled and doubled in the quiescent and beating cells during 15-day cultures. However, Ito in beating cultured cells made less contribution to APD in contrast to the quiescent cultured and freshly isolated myocytes. These findings demonstrate that electrophysiological properties differ between two types of long-term cultured cells. ICa densities remained constant in the beating cultures, suggesting that cell beating may be required for the maintenance of ICa density in developing cardiomyocytes.
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Jee, Byung Chul, Jun Woo Jo, Jung Ryeol Lee, Chang Suk Suh, Seok Hyun Kim, and Shin Yong Moon. "Effect of trichostatin A on fertilization and embryo development during extended culture of mouse oocyte." Zygote 20, no. 1 (January 27, 2011): 27–32. http://dx.doi.org/10.1017/s0967199410000547.
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SummaryWe performed this study to investigate the effect of histone deacetylase inhibition during extended culture of in vitro matured mouse oocytes. In vitro matured mouse (BDF1) oocytes were cultured in vitro for 6, 12, and 24 h, respectively, and then inseminated. During in vitro culture for 6 and 12 h, two doses of trichostatin A (TSA), a histone deacetylase inhibitor, were added (100 nM and 500 nM) to the culture medium and the oocytes were then inseminated. During the 24-h in vitro culture, two doses of TSA were added (100 nM and 500 nM) to the medium and the oocytes were activated with 10 mM SrCl2. After the 6-h culture, the fertilization rate was similar to that of the control group, but the blastocyst formation rate was significantly decreased. After the 12-h culture, both the fertilization and blastocyst formation rates were significantly decreased. After the 24-h culture, total fertilization failure occurred. In the oocytes cultured for 6 and 12 h, the fertilization and blastocyst formation rates did not differ between the TSA-supplemented and control groups. Although extended culture of the mouse oocytes significantly affected their fertilization and embryo development, TSA supplementation did not overcome their decreased developmental potential.
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Vermorken, A. J. M. "Culture Techniques and Potential Research Applications of Human Hair Follicle Cells In Vitro." Alternatives to Laboratory Animals 13, no. 1 (September 1985): 8–37. http://dx.doi.org/10.1177/026119298501300104.
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Since 1980, when human hair follicle cells were cultured in vitro for the first time, a whole series of techniques have been developed that render hair follicle keratinocytes as easy to handle in culture as fibroblasts. As a consequence, one can conclude that the need for a method providing for the routine cultivation of easily obtainable human primary epithelial cells has now been met, and it may be expected that more and more workers will use hair follicle keratinocytes for studies that specifically require human epithelial cells. The ease of culture and the ready availability of material may encourage workers to consider human hair follicle cell culture before either animal models or cultures of cells derived from invasive skin biopsies.
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Turner, K., AW Rogers, and EA Lenton. "Effect of culture in vitro and organ culture on the dry mass of preimplantation mouse embryos." Reproduction, Fertility and Development 6, no. 2 (1994): 229. http://dx.doi.org/10.1071/rd9940229.
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The dry mass of mouse embryos cultured in vitro in medium alone or in an organ culture system were measured by means of the Vickers M86 scanning microinterferometer. The data were compared with previous data on the dry mass of preimplantation embryos in vivo. The metabolism of embryos cultured in vitro differs from that of fresh embryos. In cultured embryos, dry mass decreases throughout the 2-cell stage whereas the dry mass is increasing at this stage in vivo. Embryos in an organ culture system regain a dry mass profile, similar to that observed in vivo at the late cleavage stage. These results support the view that conditions for embryo metabolism are suboptimal in vitro and that, although the oviduct may confer some advantage on developing embryos in vitro, it is unable fully to support the pattern of metabolism, as assessed by dry mass, observed in vivo.
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Jia, Zhidong, Yuan Cheng, Xinan Jiang, Chengyan Zhang, Gaoshang Wang, Jiecheng Xu, Yang Li, Qing Peng, and Yi Gao. "3D Culture System for Liver Tissue Mimicking Hepatic Plates for Improvement of Human Hepatocyte (C3A) Function and Polarity." BioMed Research International 2020 (March 4, 2020): 1–22. http://dx.doi.org/10.1155/2020/6354183.
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In vitro 3D hepatocyte culture constitutes a core aspect of liver tissue engineering. However, conventional 3D cultures are unable to maintain hepatocyte polarity, functional phenotype, or viability. Here, we employed microfluidic chip technology combined with natural alginate hydrogels to construct 3D liver tissues mimicking hepatic plates. We comprehensively evaluated cultured hepatocyte viability, function, and polarity. Transcriptome sequencing was used to analyze changes in hepatocyte polarity pathways. The data indicate that, as culture duration increases, the viability, function, polarity, mRNA expression, and ultrastructure of the hepatic plate mimetic 3D hepatocytes are enhanced. Furthermore, hepatic plate mimetic 3D cultures can promote changes in the bile secretion pathway via effector mechanisms associated with nuclear receptors, bile uptake, and efflux transporters. This study provides a scientific basis and strong evidence for the physiological structures of bionic livers prepared using 3D cultures. The systems and cultured liver tissues described here may serve as a better in vitro 3D culture platform and basic unit for varied applications, including drug development, hepatocyte polarity research, bioartificial liver bioreactor design, and tissue and organ construction for liver tissue engineering or cholestatic liver injury.
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Tähti, Hanna, Heidi Nevala, and Tarja Toimela. "Refining In Vitro Neurotoxicity Testing — The Development of Blood–Brain Barrier Models." Alternatives to Laboratory Animals 31, no. 3 (May 2003): 273–76. http://dx.doi.org/10.1177/026119290303100309.
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The purpose of this paper is to review the current state of development of advanced in vitro blood–brain barrier (BBB) models. The BBB is a special capillary bed that separates the blood from the central nervous system (CNS) parenchyma. Astrocytes maintain the integrity of the BBB, and, without astrocytic contacts, isolated brain capillary endothelial cells in culture lose their barrier characteristics. Therefore, when developing in vitro BBB models, it is important to add astrocytic factors into the culture system. Recently, novel filter techniques and co-culture methods have made it possible to develop models which resemble the in vivo functions of the BBB in an effective way. With a BBB model, kinetic factors can be added into the in vitro batteries used for evaluating the neurotoxic potential of chemicals. The in vitro BBB model also represents a useful tool for the in vitro prediction of the BBB permeability of drugs, and offers the possibility to scan a large number of drugs for their potential to enter the CNS. Cultured monolayers of brain endothelial cell lines or selected epithelial cell lines, combined with astrocyte and neuron cultures, form a novel three-dimensional technique for the screening of neurotoxic compounds.
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Rehder, Jussara, Luís Ricardo Martinhão Souto, Cláudia Maria Bernardino Magro Issa, and Maria Beatriz Puzzi. "Model of human epidermis reconstructed in vitro with keratinocytes and melanocytes on dead de-epidermized human dermis." Sao Paulo Medical Journal 122, no. 1 (February 2004): 22–25. http://dx.doi.org/10.1590/s1516-31802004000100006.
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CONTEXT: Recent progress in the field of epithelial culture techniques has allowed the development of culture systems in which the reconstructed epidermis presents characteristics of morphological differentiation similar to those seen in vivo. Human epidermis reconstructed in vitro may be used as the best alternative for the in vitro testing of the toxicology and efficiency of products for topical use, as well as in the treatment of skin burns and chronic skin ulcers. OBJECTIVE: To demonstrate a method for obtaining human epidermis reconstructed in vitro, using keratinocytes and melanocytes cultivated on dead de-epidermized human dermis. TYPE OF STUDY: Experimental/laboratory. SETTING: Skin Cell Culture Laboratory of the Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil. PROCEDURE: Human keratinocytes and melanocytes cultured in vitro were grown on a biological matrix (dead de-epidermized human dermis) and the system was kept at an air-liquid interface, in a suitable culturing medium, until a stratified human epidermis was formed, maintaining the histological characteristics of the epidermis in vivo. RESULTS: It was histologically demonstrated that it is possible to reproduce a differentiated epidermis through keratinocytes and melanocytes cultured on dead de-epidermized human dermis, thus obtaining a correctly positioned human epidermis reconstructed in vitro with functional keratinocytes and melanocytes that is similar to in vivo epidermis. CONCLUSIONS: It is possible to obtain a completely differentiated human epidermis reconstructed in vitro from keratinocyte and melanocyte cultures on a dead de-epidermized human dermis.
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Reed, Barbara M. "COLD STORAGE OF IN VITRO RUBUS GERMPLASM." HortScience 27, no. 6 (June 1992): 695f—695. http://dx.doi.org/10.21273/hortsci.27.6.695f.
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In vitro cold storage of Rubus germplasm was investigated using several environmental conditons and types of storage containers. Shoot cultures of Rubus species and cultivars were grown in either tissue culture bags or 20 × 150 mm glass tubes and compared for plant condition and survival under various storage conditions. Cultures stored at 10 C in the dark were in poor condition after 6 months. Cultures kept at 4 C were in much better condition and had higher survival rates after 18 months when stored with a 12 h daylength rather than total darkness. Overall there were no differences in survival or condition between cultures in tubes and bags. Contamination rates were 15% in tubes and 0% in bags. Plants in tissue culture bags could be stored for 9 months at 25 C with 16 h light when the nitrogen level of the MS medium was reduced to 25% and the medium volume was increased from 10 to 20 ml per bag. Genotype differences were apparent under all conditions tested. The best storage condition for Rubus germplasm was 4 C with 12 h light. Plastic tissue culture bags were preferred over tubes due to lower contamination rates.
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Corrêa da Silva, Diogo Pedrosa, Elif Aylin Ozudogru, Michele Valquíria Dos Reis, and Maurizio Lambardi. "In vitro conservation of ornamental plants." Ornamental Horticulture 24, no. 1 (March 12, 2018): 28–33. http://dx.doi.org/10.14295/oh.v24i1.1163.
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The market of flowers and ornamental plants is dependent on the diversification of species and the availability of high quality propagation materials. Actually, in vitro culture techniques performance a prominent role in the multiplication and maintenance of commercially propagated ornamental plant species, and are promising for the production of thousands of high quality plants in relatively short term. In addition, when market demand for a particular species is low or zero in a specific period of the year, in vitro culture techniques allow the conservation of cultures under aseptic conditions, by Slow Growth Storage (SGS), from a few weeks to one year (or more), without affecting their viability and potential regrowth. This can be achieved by modifying the constitution of the culture medium and the maintenance conditions of in vitro cultures. Obviously, the success of the technique depends on greatly on the physiological characteristics of the species to be conserved, as well. Once a SGS protocol is optimized, the expenses labor, the possibility of contamination and the probability of somaclonal variation can be reduced markedly.
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Do, Giap Dang, Hien Thi Dieu Huynh, The Danh Tran, and Tuan Trong Tran. "USING NATURAL LIGHT ON MICROPROPAGATION OF SWEET POTATO ( Ipomoea batatas L. ) IN AREA OF HO CHI MINH CITY." Science and Technology Development Journal 15, no. 3 (September 30, 2012): 36–46. http://dx.doi.org/10.32508/stdj.v15i3.1845.
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Plantlets of sweet potato ( Ipomoea batatas L. ) were cultured in vitro under three different ambient conditions including a standard culture room - PS, a culture room inside a glasshouse with natural light but controlled temperature - TH, and a standard glasshouse with natural light (natural fluctuations of temperature) - NP. Plantlets from three treatments were compared in terms of pathogen rate, growth, survival plant at the end of the in vitro stage and at the ex vitro acclimatization. This result showed that, after 28 days of culture, sweet potato plants were cultured in vitro TH conditions have reduced entirely due to susceptibility to fungal disease causing outside air. After 14 days of ex vitro acclimatization, plants originally grow in vitro under the TH condition had ability to adapt about field survival and growth rates better than the other two treatments.
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Shannon, J. M., S. D. Jennings, and L. D. Nielsen. "Modulation of alveolar type II cell differentiated function in vitro." American Journal of Physiology-Lung Cellular and Molecular Physiology 262, no. 4 (April 1, 1992): L427—L436. http://dx.doi.org/10.1152/ajplung.1992.262.4.l427.
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We have investigated whether the loss of differentiated function observed in adult rat alveolar type II cells cultured on a substratum that promotes cell spreading and flattening represents a reversible phenotypic change. Cells were cultured for 4 and 8 days in association with fetal rat lung fibroblast feeder layers on either attached collagen gels, which promote the loss of differentiated function, or on floating collagen gels, which support differentiation. A fifth group of cultures were maintained as attached gels for 4 days, then detached and cultured as floating gels for the remaining 4 days. Expression of mRNAs for surfactant proteins A, B, and C, patterns of phospholipid biosynthesis, rates and patterns of protein synthesis, and cell morphology were evaluated as markers of differentiation. Without exception, detaching the gels after 4 days in culture resulted in significant recovery of differentiated characteristics, demonstrating that type II cells modulate differentiated function in response to the culture environment. The results are discussed in relation to the importance of normal cell architecture to normal cell function and to the possible in vitro progression of type II cells to type I cells.
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Thomas, Philip T., and Patrick T. K. Woo. "In vitro culture and multiplication of Cryptobia catostomi and experimental infection of white sucker (Catostomus commersoni)." Canadian Journal of Zoology 70, no. 2 (February 1, 1992): 201–4. http://dx.doi.org/10.1139/z92-031.
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Cryptobia catostomi, a parasitic haemoflagellate of the white sucker (Catostomus commersoni), was cultured in minimum essential medium (MEM) supplemented with Hanks' salts, L-glutamine, and 25% foetal bovine serum (MEM-plus). Parasite numbers were significantly higher in MEM-plus cultures supplemented with white sucker plasma than in unsupplemented cultures. This procedure is useful when large numbers of the parasite are required, e.g., for studies on their nutritional requirements, metabolism, or antigenic nature. Cultures could not be maintained at 10 °C beyond the fourth subculture; this was about 11 months after the primary culture was started. The division process in culture was similar to that reported in fish. The culture forms were infective to white suckers. Parasitaemias in white suckers infected with blood forms increased from 2 to 5 weeks postinfection and stayed relatively constant thereafter. Neither anorexia nor anaemia was evident in infected fish, confirming the nonpathogenicity of C. catostomi to white suckers.
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Silva, G. M., R. Rossetto, R. N. Chaves, A. B. G. Duarte, V. R. Araújo, C. Feltrin, M. P. Bernuci, et al. "In vitro development of secondary follicles from pre-pubertal and adult goats cultured in two-dimensional or three-dimensional systems." Zygote 23, no. 4 (March 26, 2014): 475–84. http://dx.doi.org/10.1017/s0967199414000070.
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SummaryThe aim of this study was to evaluate the influence of two-dimensional (2D) and three-dimensional (3D) alginate culture systems on in vitro development of pre-antral caprine follicles. In addition, the influence of the reproductive age of the ovary donor on the in vitro culture success was investigated. Pre-antral follicles from pre-pubertal or adult goats were isolated and cultured directly on a plastic surface (2D) or encapsulated in an alginate-based matrix (3D). After 18 days, the oocytes underwent in vitro maturation (IVM) and in vitro fertilization (IVF) to produce embryos. The 3D system showed higher rates of follicle survival, lower rates of oocyte extrusion, and a greater number of recovered oocytes for IVM and IVF (P < 0.05). Only pre-antral follicles from adult animals produced MII oocytes and embryos. The estradiol concentrations increased from day 2 to day 12 of culture in all groups tested (P < 0.05). Conversely, progesterone concentrations were lower in 3D-cultured follicles than in 2D-cultured follicles, with differences on days 2 and 6 of culture (P < 0.05). We provide compelling evidence that a 2D or 3D alginate in vitro culture system offers a promising approach to achieving full in vitro development of caprine pre-antral follicles to produce mature oocytes that are capable of fertilization and viable embryos.
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Bonamico, Margherita, Luigi Sabbatella, Marco Di Tola, Stefania Vetrano, Mirella Ferri, Raffaella Nenna, Paolo Mariani, and Antonio Picarelli. "Antiendomysial Antibody Detection in Biopsy Culture Allows Avoidance of Gluten Challenge in Celiac Children." Journal of Pediatric Gastroenterology and Nutrition 40, no. 2 (February 2005): 165–69. http://dx.doi.org/10.1002/j.1536-4801.2005.tb00957.x.
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ABSTRACTObjective:Antiendomysial antibody (EMA) production has been induced in vitro by the small bowel mucosa of celiac disease (CD) patients in clinical remission cultured in the presence of gliadin peptides. The aim of the present study was to use this in vitro system to determine whether it could be used to predict the clinical or histologic relapse to gluten challenge in CD children on a gluten‐free diet (GFD).Methods:Enrolled were 32 CD children and adolescents on GFD (group 1), and 80 controls (group 2) who underwent in vitro gliadin challenge. Subsequently, 24 group 1 CD children underwent in vivo gluten challenge to confirm the diagnosis. Biopsy cultures, with and without gliadin, morphometric analysis, immunoglobulin (Ig)A and IgG1 EMA detection, both in sera and culture supernatants, were performed.Results:Of the 32 group 1 CD patients, 23 were IgA EMA positive in culture supernatants. The other nine were IgG1 EMA positive. All 24 children who had in vivo gluten challenge showed clinical or histologic relapse. All culture supernatants from disease controls belonging to group 2 were both IgA and IgG1 EMA negative, irrespective of gliadin challenge.Conclusions:Organ culture with in vitro gliadin challenge is able to reproduce the results of in vivo challenge. This system could reduce the need for gluten challenge in celiac children.
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Redel, B. K., L. D. Spate, A. N. Brown, and R. S. Prather. "97 SUPPLEMENTATION WITH FOLATE IN VITRO INCREASES TROPHECTODERM AND TOTAL CELL NUMBER IN IN VITRO-DERIVED PORCINE BLASTOCYSTS." Reproduction, Fertility and Development 24, no. 1 (2012): 161. http://dx.doi.org/10.1071/rdv24n1ab97.
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It is vital that improvements are made to current culture environments because in vitro culture systems are suboptimal compared with in vivo. A previous transcriptional profiling endeavour conducted by Bauer et al. (2010 Biol. Reprod. 83, 791–798) identified hundreds of mRNA transcripts that were mis-expressed in porcine embryos fertilized in vivo and then cultured in vitro to Day 6 compared with in vivo Day-6 embryos. Enriched in the downregulated transcripts were 4 genes involved with the one carbon pool by folate KEGG pathway. This downregulation of genes involved with folate metabolism may illustrate an impaired folate homeostasis in embryos cultured in the current culture environment. The objective of this study was to determine the effects folate had on embryo development of in vitro fertilized embryos. Porcine cumulus–oocyte complexes were matured for 44 h in M199 supplemented with epidermal growth factor (EGF), FSH and LH. Oocytes with a visible polar body were selected and fertilized in modified tris buffered medium for 5 h and then placed into porcine zygote medium 3 with 0 mM, 0.2 mM, 0.4 mM and 0.8 mM folate to find the optimal concentration of folate. Twenty-eight hours post-fertilization, cleaved embryos were selected and moved into 25-μL drops of respective culture medium and cultured to Day 6 in a water-saturated atmosphere of 5% CO2, 5% O2, 90% N2, at 38.5°C. To determine the effect folate had on development, the blastocyst rate for each treatment group was measured. Results were log-transformed and analysed by using PROC GLM in SAS (SAS Institute Inc., Cary, NC). A least-significant difference post-test comparison was completed to determine if significant differences existed between treatment groups. The percentage of cleaved embryos on Day 6 that developed to blastocyst was 56.2%, 55.9%, 66.9% and 61.8% (n = 133, 149, 135 and 135) in 0 mM, 0.2 mM folate, 0.4 mM folate and 0.8 mM, respectively. The 0.4 mM folate group tended (P = 0.07) to have a higher number of cleaved embryos that developed to the blastocyst stage. Consequently, this concentration was used for all further embryo culture experiments. Differential staining was completed to compare the number of trophectoderm and inner cell mass nuclei for embryos cultured in 0 mM or 0.4 mM folate concentrations. Staining revealed that embryos cultured with folate had an increase in number of trophectoderm (29.7 ± 1.5 vs 24.4 ± 1.4 cells; P = 0.0058) and total cell (36.9 ± 1.0 vs 31.7 ± 1.0; P = 0.0007) numbers compared with embryos cultured without folate. These results illustrate that the addition of folate to current culture medium doesn't hinder development to blastocyst and by increasing trophectoderm and total cell number may give rise to better-quality in vitro-derived embryos. It is evident that using transcriptional profiling can be a great method of identifying ways to improve embryo culture systems and, in this case, supplementing with folate. Funded by Food for the 21st Century.
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Obata, Y., and T. Kono. "254 DEVELOPMENTAL COMPETENCE OF MOUSE OOCYTES AFTER IN VITRO GROWTH, NUCLEAR TRANSFER, AND IN VITRO FERTILIZATION." Reproduction, Fertility and Development 19, no. 1 (2007): 243. http://dx.doi.org/10.1071/rdv19n1ab254.
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Long-term effects of in vitro maturation of oocytes and in vitro culture of fertilized eggs have been reported in ruminants, mice, and humans. However, effects of in vitro oocyte growth are unknown. Although a large number of non-growing oocytes can be a gamete resource, very few oocytes ever acquire competence to support full-term development after in vitro growth. The objective of the study was to evaluate different culture conditions and the long-term effects of in vitro oocyte growth on the production of offspring. Oocytes of newborn, 10-day-old, and adult BDF1 (C57BL/6N � DBA2) mice were cultured for 22, 11, and 1 day(s), respectively. The results showed that alpha-MEM medium was superior to Waymouth medium in oocyte growth (68.6 � 3.87 �m vs. 61.7 � 3.26 �m, respectively; P < 0.001), and in maintenance of follicular integrity (69% vs. 30%; P < 0.001) when non-growing oocytes from newborn mice were cultured. However, oocytes grown in vitro were incompetent to support meiotic maturation by themselves in the case of either the 22-day culture of oocytes from newborn mice (1/59 in alpha-MEM vs. 1/65 in Waymouth) or the 11-day culture of oocytes from 10-day-old mice (51/140 in alpha-MEM vs. 2/157 in Waymouth), and none of them developed to the blastocyst stage. Subsequently, to examine the nucleic competence of oocytes grown in vitro, serial nuclear transfers were carried out. Karyoplasts from oocytes grown in vitro using alpha-MEM were fused with the GV oocytes grown in vivo after enucleation. The reconstituted oocytes were cultured in alpha-MEM. After 14 h, MII chromosomes of the reconstituted oocytes were transferred into the enucleated and ovulated MII oocytes in order to provide cytoplasmic competency. The results showed that when the donor oocytes attained a diameter of e60 �m, the reconstituted oocytes could develop into pups at extremely high rates (30-41%) after in vitro fertilization (IVF) and embryo transfer in the case of either the 22-day culture of oocytes from newborn mice (7/17) or the 11-day culture of oocytes from 10-day-old mice (25/77). A significant difference was not observed in the competence to develop to term of the reconstituted oocytes when compared with that of the oocytes reconstituted from the control GV (25/52; P > 0.05). When the donor oocytes attained a diameter of 50–60 �m, the reconstituted oocytes also could develop into pups (7/33); however, their efficiency was significantly reduced when compared with that of the reconstituted oocytes from the control GV (P < 0.05). On the other hand, the weight of the offspring depended on the duration of culture, and offspring from non-growing oocytes (1.48 � 0.17 g) were heavier than those of the IVF control (1.25 � 0.14 g; P < 0.05). In conclusion, we have demonstrated that using a nuclear transfer technique combined with in vitro growth of oocytes was sufficient to produce functional oocytes, and long-term culture for oocyte growth did not affect the nucleic ability of oocytes to develop to term; however, fetal growth may be susceptible to the duration of culture.
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Cott, G. R., J. Y. Westcott, and N. F. Voelkel. "Prostaglandin and leukotriene production by alveolar type II cells in vitro." American Journal of Physiology-Lung Cellular and Molecular Physiology 258, no. 4 (April 1, 1990): L179—L187. http://dx.doi.org/10.1152/ajplung.1990.258.4.l179.
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Alveolar type II cells were isolated from adult rats, cultured for 22 h, and individual eicosanoids in the media from unstimulated and stimulated cells were quantified by immunoassay. Stimulation with the calcium ionophore A23187 significantly increased the media levels of prostaglandins (prostaglandin and 6-keto-prostaglandin F1 alpha greater than thromboxane B2). In contrast to previous reports, increased media levels of leukotrienes were also recovered from cells incubated with A23187, but only for cells in culture for less than or equal to 24 h. The production of leukotriene C4 was confirmed by a combination of high-performance liquid chromatography and spectrophotometric analysis. The profile of eicosanoids produced by cultures of alveolar type II cells was distinctly different than that of similarly cultured alveolar macrophages. Finally, stimulation of alveolar type II cell cultures with either a phorbol ester or phospholipase C increased media prostaglandin levels but failed to increase leukotriene levels. We conclude that primary cultures of alveolar type II cells are capable of the de novo metabolism of arachidonic acid to both cyclooxygenase and lipoxygenase products and that the production of leukotrienes is dependent on both time in culture and agonist. Thus alveolar type II cells are a potential source for the production of these eicosanoids in vivo, and the particular lipid mediators produced may vary depending on the pathophysiologic stimulus.
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Nas, Mehmet Nuri, Nedim Mutlu, and Paul E. Read. "Random Amplified Polymorphic DNA (RAPD) Analysis of Long-term Cultured Hybrid Hazelnut." HortScience 39, no. 5 (August 2004): 1079–82. http://dx.doi.org/10.21273/hortsci.39.5.1079.
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RAPD and phenotypic analysis were conducted to assess clonal stability of hazelnuts generated from axillary buds cultured in vitro for long-term. The nuts produced on in vitro-propagated plants were indistinguishable from those of donor plants. With the exception of rare horizontal (plagiotropic) growth, all in vitro-propagated plants exhibited phenotypes similar to those of donor plants. RAPD analysis did not reveal any somaclonal variation between donor plants from which in vitro cultures were initiated and micropropagated plants (6-year cultures), and no somaclonal variation was detected among in vitro-propagated plants. However, polymorphism (15.6%) was detected between the parent plant and its in vitro-propagated progenies (from seedlings). These results show a good discriminatory power of RAPD to detect polymorphism between samples where it is expected, and it can be effectively used for genetic assessment of micropropagated hazelnut. No evidence of genetic or epigenetic changes was observed in long-term cultured hazelnut, and thus long-term in vitro culture of hazelnut does not seem to limit its clonal propagation.
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Palla, Kaitlin J., Rochelle R. Beasley, and Paula M. Pijut. "In Vitro Culture and Rooting of Diospyros virginiana L." HortScience 48, no. 6 (June 2013): 747–49. http://dx.doi.org/10.21273/hortsci.48.6.747.
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The hard, strong, very close-grained wood of common persimmon (Diospyros virginiana L.; Ebenaceae) is desirable for specialty products such as golf club heads, percussion sticks, billiard cues, and for wood turnery. The edible fruit of cultivated varieties is sold as pulp for use in puddings, cookies, cakes, and custards. Persimmon is usually propagated by grafting. Own-rooted clonal persimmon could offer several advantages to specialty fruit growers such as elimination of grafting, graft incompatibility issues, and improved rootstocks for variety testing. Four mature, grafted (male and female) persimmon genotypes and one hybrid were used for nodal explant culture. Nodal stem explants were cultured on Murashige and Skoog (MS) medium containing 10 μM zeatin, 3% (w/v) sucrose, and 0.7% (w/v) Bacto agar. Explants were routinely transferred to fresh medium every 3 weeks until shoot cultures were established. All nodal explants excised from grafted greenhouse plants produced at least one viable shoot. For in vitro rooting of microshoots, half-strength MS medium with 0, 5, 10, or 15 μM indole-3-butyric acid (IBA), 0.1 g·L−1 phloroglucinol, 3% (w/v) sucrose, and 0.7% (w/v) Bacto agar were tested with a 10-day dark culture treatment followed by culture in the light. Best rooting (14% to 87%) was achieved on medium containing 5 μM IBA for the common persimmon genotypes with means averaging from 0.5 to 3.9 roots per shoot. Ninety-one percent rooting with 5.3 ± 2.6 roots per shoot was achieved for the hybrid persimmon. Rooted plants were successfully acclimatized to the greenhouse.