The application of blastomere separation (BS) technique to produce embryonic stem cell (ESC) from inner cell mass (ICM) is not yet perfected due to low cleavage efficiency of single blastomere. Thefore, the aims of this study were to evaluate the developmental potential of single blastomere (SB) derived from embryos at different stages to blastocyst and the potential of ICM outgrowth. The female mice of ICR strain (age: 8-12 weeks; n=47) were superovulated using gonadotrophins and mated with ICR strain males (age: 10-14 weeks; n=20). The 2-cell embryos (n=366) were flushed from the oviduct of the treated females and cultured in vitro before assigned into the following groups: a) 2-cell, b) 4-cell, c) 8-cell and d) control prior blastomere separation. The SBs were cultured in vitro and daily observations were conducted to record the cleavage rates up to Day 5. Developmental rate of 2-cell derived SB (77.28±6.77) was greater than 4-cell (63.70±5.35) and 8-cell (55.73±3.35), corresponding to the results of ICM outgrowth at 2-cell (69.29±4.13), gave higher rate followed by 4-cell (55.73±7.81) and 8-cell (41.85±3.58). Diameter of blastocyst decreased as the SB parent embryo stage increased, with the respective ratio of 5 (diameter of SB blastocyst): 3 (diameter of SB) (2-cell: 92.55±1.59 vs. 56.48±0.40; 4-cell: 78.71±1.37 vs. 44.02±0.49 and 8-cell: 64.13±2.20 vs. 35.68±0.34) as well as total cell number in blastocyst (2-cell: 43.00±1.48; 4-cell: 28.33±1.15; 8-cell: 8.80±0.58). In conclusion, SB at different stages of mouse embryos successfully develop to blastocysts in vitro that can be used as ICM source, which is a prerequisite for establishment of ESC outgrowth.
The inconsistency of efficiency in murine embryonic stem cell (ESC) production might be associated with the differences in preparation and cryopreservation of the feeder cell layer. As the cryopreservation of mouse embryonic fibroblast (MEF) declined the quality of MEF as feeder cell layer, an effective protocol should be determined to produce murine ESC on frozen-thawed feeder cell layer as efficient on fresh feeder cell layer. Under appropriate culture conditions, isolated inner cell mass (ICM) of murine blastocyst will form ESC and be maintained in undifferentiated state. Therefore, the aims of this study were to determine the most optimum freezing density and equilibration duration for cryopreserving MEF feeder cell layer and to determine the effect of fresh and frozen-thawed feeder cell layer on murine ESC production. Freezing density of 5×106 cells÷mL gave a significantly higher viability rate than 0.5×106 cells÷mL (68.08% vs. 59.78%, p<0.05) and comparable with 2×106 cells÷mL. The viability rates of frozen-thawed MEF derived from 15 minutes equilibration was significantly higher than 20 hours equilibration (79.4% vs. 68.08, p<0.05). There were no significant differences between fresh and frozen-thawed MEF feeder cell layer for percent successful attachment of blastocysts, consecutive passages of murine ESC up to passage 3. In conclusion, freezing density of 5×106 cells÷mL and 15 minutes equilibration duration are optimizing the cryopreservation of MEF feeder cell layer to subsequently improve the production of murine ESC.