In vitro embryos production after oocytes treatment with forskolin.

The inhibition of nuclear maturation allows time for the oocyte to accumulate molecules that are important for embryonic development. Thus, the objective of this work was to evaluate the effect of blocking oocyte meiosis with the addition of forskolin, an efficient inhibitor of nuclear maturation, in in vitro maturation (IVM) medium. Forskolin was added to the IVM medium for 6 h at concentrations of 0.1 mM, 0.05 mM or 0.025 mM, then the oocytes were allowed to mature in drug-free medium for 18 h. The oocytes were assessed for the stage of nuclear maturation, the activity and distribution of mitochondria, oocyte ultrastructure, the number of viable cells and the apoptosis rate. After forskolin treatment, the oocytes were fertilized in vitro and cultured for 7 days. On day 7, the blastocyst rate, the ultrastructure, the number of intact cells and the apoptosis rate of the blastocysts were measured. No differences were observed for the stage of nuclear maturation of the oocyte, the mitochondrial activity and distribution, the blastocyst rate or total number of intact cells. However, a higher rate of apoptosis was observed in the blastocysts produced from oocytes blocked for 6 h with the higher concentration of forskolin (P < 0.05). We conclude that all the experimental groups reached the MII stage after the addition of forskolin and that the highest concentration of forskolin caused cellular degeneration without harming embryo production on the 7th day.

Evaluation of castor oil-based polyurethane membranes in rat bone-marrow cell culture.

PURPOSE: To evaluate three methods to isolate rats MSCs and to analyze the potential of a castor oil polyurethane base membrane as a scaffold for MSCs. METHODS: Four male Wistar rats, aged 20-30 days were used. Bone marrow aspirates from femur and tibia were harvested using DMEM high glucose and heparin. The cell culture was performed in three different ways: direct culture and two types of density gradients. After 15 days, was made the 1st passage and analyzed cell viability with markers Hoerscht 33342 and propidium iodide. The MSCs were characterized by surface markers with the aid of flow cytometry. After this, three types of castor oil polyurethane membranes associated with the MSCs were kept on the 6-well plate for 5 days and were analyzed by optical microscopy to confirm cell aggregation and growth. RESULTS: Separation procedures 1 and 2 allowed adequate isolation of MSCs and favored cell growth with the passage being carried out at 70% confluence after 15 days in culture. The cells could not be isolated using procedure 3. When the 3 castor oil polyurethane membrane types were compared it was possible to observe that the growth of MSCs was around 80% in membrane type 3, 20% in type 2, and 10% in type 1. CONCLUSION: Both Ficoll-Hypaque densities allow isolation of rat MSCs, and especially castor oil-based membrane type 3 may be used as a scaffold for MSCs.

Evaluation of castor oil-based polyurethane membranes in rat bone-marrow cell culturey / Avaliação de membranas à base de óleo de polímero de mamona em culturas de células de medula óssea de ratos

PURPOSE: To evaluate three methods to isolate rats MSCs and to analyze the potential of a castor oil polyurethane base membrane as a scaffold for MSCs. METHODS: Four male Wistar rats, aged 20-30 days were used. Bone marrow aspirates from femur and tibia were harvested using DMEM high glucose and heparin. The cell culture was performed in three different ways: direct culture and two types of density gradients. After 15 days, was made the 1st passage and analyzed cell viability with markers Hoerscht 33342 and propidium iodide. The MSCs were characterized by surface markers with the aid of flow cytometry. After this, three types of castor oil polyurethane membranes associated with the MSCs were kept on the 6-well plate for 5 days and were analyzed by optical microscopy to confirm cell aggregation and growth. RESULTS: Separation procedures 1 and 2 allowed adequate isolation of MSCs and favored cell growth with the passage being carried out at 70 percent confluence after 15 days in culture. The cells could not be isolated using procedure 3. When the 3 castor oil polyurethane membrane types were compared it was possible to observe that the growth of MSCs was around 80 percent in membrane type 3, 20 percent in type 2, and 10 percent in type 1. CONCLUSION: Both Ficoll-Hypaque densities allow isolation of rat MSCs, and especially castor oil-based membrane type 3 may be used as a scaffold for MSCs.

OBJETIVO: Avaliar três formas de cultivo de células-tronco mesenquimais de ratos; e analisar o potencial do polímero de mamona na forma de membrana como arcabouço para CTMs. MÉTODOS: Foram utilizados quatro ratos machos Wistar, de 20 a 30 dias de idade. Aspirados da medula óssea do fêmur e da tíbia foram colhidos com DMEM alta glicose e heparina. As células foram isoladas de três formas diferentes: cultivo direto e com dois tipos de gradientes de densidade. Após 15 dias, foi feita a 1ª passagem e analisada a viabilidade celular com os marcadores Hoerscht 33342 e Iodeto de Propídio. As CTMs foram então caracterizadas por marcadores de superfície, com o auxílio de citômetro de fluxo. Após, três tipos de membrana à base de óleo de polímero de mamona associadas com as CTMS foram mantidas em cultivo por cinco dias, e analisados por microscópio ótico para confirmar o crescimento e a adesão celular. RESULTADOS: Após 15 dias, Os procedimentos que utilizaram gradientes de densidade permitiram o isolamento das CTMs e favoreceram o crescimento celular com a passagem, sendo obtido 70 por cento de confluência após 15 dias em cultura. O procedimento direto não se mostrou eficaz para o isolamento das células. O crescimento das CTMs foi aproximadamente 80 por cento sobre a membrana tipo 3, 20 por cento na tipo 2 e 10 por cento na membrana tipo 1. CONCLUSÃO: Os dois gradientes de concentração Ficoll-Hypaque permitem isolar CTMs de ratos; e especialmente a membrana de polímero de mamona tipo 3 pode ser usada como um bom arcabouço para as CTMs.