Effectiveness of microwell-based in vitro culture systems for zona-free cloned bovine embryos
Feltrin, CristianoForell, FabianaMachado, MaluQueiroz, Luis MauroPeixer, MaurícioMalard, PatríciaSantana, GilmarWheeler, Matthew BenjaminRodrigues, José Luiz
Background: Modified embryo in vitro culture (IVC) systems have been devised for the culture of individual embryos, suchas in microdroplets, capillary tubes, or co-cultures systems. However, the development of the Well-of-the-Well (WOW)system takes advantage of both the individual and the grouped culture systems, as embryos are cultured into microwellsdisposed into a larger well. The aim of this study was to evaluate the in vitro and in vivo developmental potential of handmade cloning (HMC)-derived cloned bovine embryos after the IVC in three microwell (WOW) systems.Material, Methods & Results: Adipose tissue-derived mesenchymal stem cells (MSCs) isolated after subcutaneous biopsy from a Nellore adult female were used as nucleus donor cells for cloning. Collected adipose tissue was incubated in0.075% collagenase for 60 min at 39oC. After digestion, cell suspension was centrifuged at 80 g for 10 min, supernatantwas discarded, and 2 mL of Red Blood Cells Lysis Buffer (RBC) was added to the pellet, remaining for 2 min. The RBCbuffer was diluted with 20 mL PBS, with cell suspension spun at 80 g for 5 min. Supernatant was discarded and the pelletre-suspended in DMEM culture medium + 10% fetal calf serum (FCS). Following cell counting, 5 x 105 cells were seededin 25-cm² bottles containing 6 mL DMEM + 10% FCS, and cultured at 38.3°C, 5% CO2 in air and saturated humidity.After IVM, oocytes were denuded and incubated in demecolcine, followed by zona pellucida removal, oocyte bisection,embryo reconstruction, electrofusion and chemical activation. Cloned embryos were allocated to one of three IVC groups:cWOW: conventional microwells (250 µm, round); mWOW: modified microwells (130 µm, conical); and WOW-PDMS:microwells in polydimethylsiloxane chips (170 µm, cylindrical, with interconnecting microchannels); IVF embryos wereused as controls. Cleavage (D2), blastocyst (D7) and pregnancy (D30) rates were analyzed by the χ2 test, for P < 0.05...(AU)
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