Effectiveness of Microwell-Based In Vitro Culture Systems for Zona-Free Cloned Bovine Embryos

Background: Modified embryo in vitro culture (IVC) systems have been devised for the culture of individual embryos, such as 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 microwells disposed 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 in 0.075% collagenase for 60 min at 39 degrees C. After digestion, cell suspension was centrifuged at 80 g for 10 min, supernatant was discarded, and 2 mL of Red Blood Cells Lysis Buffer (RBC) was added to the pellet, remaining for 2 min. The RBC buffer was diluted with 20 mL PBS, with cell suspension spun at 80 g for 5 min. Supernatant was discarded and the pellet re-suspended in DMEM culture medium + 10% fetal calf serum (FCS). Following cell counting, 5 x 10(5) cells were seeded in 25-cm(2) bottles containing 6 mL DMEM + 10% FCS, and cultured at 38.3 degrees 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 mu m, round); mWOW: modified microwells (130 mu m, conical); and WOW-PDMS: microwells in polydimethylsiloxane chips (170 mu m, cylindrical, with interconnecting microchannels); IVF embryos were used as controls. Cleavage (D2), blastocyst (D7) and pregnancy (D30) rates were analyzed by the chi(2) test, for P < 0.05. Blastocyst rates in the IVF group (315/594; 53.0%) were higher than in cloned groups, whereas between clones, blastocyst rates in mWOW (99/239; 41.4%) were higher than in cWOW (72/232; 31.0%) and in WOW-PDMS (68/234; 29.1%). Discussion: Many in vitro embryo culture systems have been developed over the past 20 years, with a wide range of features. One of the big trends has been the development of IVC systems that could increase the in vitro and in vivo developmental potential of IVP embryos. Simple, chemically defined, semi-defined, and sequential media, or physical IVC requirements (individual embryo culture, or microchannel or microfluidic IVC systems) have been proposed as ways to attain higher rates of embryo development after culture. Nonetheless, such systems are sub-optimal, still failing to mimic the in vivo environment during early development. In this study, a custom-made microfluidic system (WOW-PDMS) was devised for the IVC of zona-free handmade cloned bovine embryos, comparing results for in vitro and in vivo embryo development with standard and modified microwell IVC systems. Overall in vitro and in vivo relative efficiency for embryo production and establishment of pregnancy, and in vivo embryo development to Day 30 of pregnancy was not different between the IVF and the cloned groups. In summary, the narrower microwell system promoted better in vitro development to zona-free HMC-derived cloned embryos than the conventional and the PDMS-based microwell systems, with no differences on in vivo development between systems.