Feeder layer-free culture of inner cell mass and trophoblastic explants obtained from bovine IVF blastocysts

BERTOLA ME; SUELDO C,; SALAMONE D

USA

Congreso; ASRM; 2006

ASRM

Objective To determine the in vitro development of inner cell mass (ICM) and trophoblastic (T) explants without feeder layers obtained from bovine IVF blastocysts. Design Descriptive in vitro study. Materials and methods Bovine ovaries were collected at local abattoir and cumulus-oocyte complex(COC) were obtained by aspiration of 2 to 5 mm follicles. Oocytes surrounded by more than 4 layers of cumulus cells were matured in 500 ul TCM 199 supplemented with 5% fetal bovine serum (FBS) and 1 mg/ml of FSH under 200 ul of mineral oil. All cultures were done at 39°C with 5% CO2 in a humidified atmosphere for 23 h. Frozen /thawed spermatozoa were washed twice with Brackett-Oliphant (BO) medium, without BSA, but supplemented with 5 mM of caffeine and 20 ug/ml of heparin. The concentration of semen was adjusted and diluted in half with B0 plus 10 mg/ml of BSA. The COC were exposed for 6 h to sperm added at a final concentration of 1 x 107 /ml. After fertilization, embryos were co-cultured with cumulus cells for 9 days.Each of the 27 blastocysts obtained was treated by mechanical microsurgery, separating the ICM from the T. The culture of the ICM and T obtained was done randomly placing 3 ICM or 3 T in 5 ul droplets of MEM media containing 20 ul of insulin, selenium and transferrin, pennicillin-streptomycin, 5% BFS and 10 ul/ml of LIF under mineral oil and incubated in 5% CO2 at 39oC temperature. To evaluate cellular growth, observations were done from 1 to 60 days at 1000X of magnification. Results After 2 days of culture all the T were clearly inert and arrested, while the ICM showed an intense adhesion and proliferation in the culture dishes and were able to cover different % of the microdrops, some of them were able to show abundant cell proliferation after 25 days and some remained vital after 2 months in culture as shown in Table 1. Full-size table View Within Article Conclusion Our data shows that the blastocyst components once separated in ICM and T differ in their needs in vitro under our culture conditions. Our culture model was able to maintain an intense proliferation of the ICM keeping stem cells morphology. The absence of feeder layer co-culture is beneficial, diminishing the exposure to potential contaminants and simplifying culture conditions. These culture conditions could be applied to culturing human embryonic explants in order to generate stem cells without the threat of interspecies cell contamination