PMC

Embryos flowing into the microfluidic culture chamber by gravity. Two 2-cell stage mouse embryos have fallen to the bottom part of the funnel (a), entered the microfluidic channel (b), to cross the open valves (c), and enter the circular, 100 nL culture chamber (d). (d) The valve was closed immediately after embryos passed it.

Microfluidic embryo culture chip design. (a) Front view. (b) Side view. (c, d) Top view (top panels) and cross-sectional view (bottom panels) of a flow channel crossing over a control channel respectively, show how a membrane valve is created. If there is no pressure difference across the PDMS membrane, the membrane valve remains open (c). When the pressure in the control channel exceeds the flow channel pressure (by approx 10 psi) the membrane will flex and close off the flow channel (d). (e) Photograph of the microfluidic chip. The flow channels are filled with dark color dye and the control channels are filled with a lighter color dye. Chip shown contains two 100 nL and two 500 nL chambers.

Experimental protocol and blastocyst development in microfluidic chip. (a) 1-cell stage embryos were harvested on Day 0, cultured in groups of 10 embryos per 20 μL drop, and transferred to 100 nL microfluidic chambers for culture in groups of 2 embryos per chamber, while a transfer control comprising 10 embryos per group was performed in parallel. The same protocol is performed to test groups of 2 embryos in conventional 5 μL and 20 μL microdrops, in parallel with transfer and non-transfer controls. (b) Blastocyst development for groups of 2 embryos was comparable between 100 nL and 20 μL, but it was significantly compromised in the 5 μL drop. Blastocyst developmental rates were above 90% for groups of 10 embryos cultured in the transfer control and non-transfer control drops, which indicated that in vitro environment was well maintained in all the experiments.

Group culture is required for in vitro mouse embryo development. (a) At 5 μL culture volume, the blastocyst development rate was 50.0 ± 8.9% for embryos cultured singly, which was lower than the rate of 90.0 ± 0.0% observed for embryos cultured in groups of 10 (p-value = 0.01). At 20 μL culture volume, the blastocyst development rate was 86.6 ± 3.3% for embryos cultured singly, which was lower than the rate of 97.0 ± 2.1% observed for embryos cultured in groups of 10 (p-value = 0.08). Paired t-tests were performed. Mean ± standard error of the mean (SEM). (b) Singly-cultured embryos that were transferred to a new culture drop resulted in decreased blastocyst developmental rates at 0.0 ± 0.0% for 5 μL and 70.5 ± 5.2% for 20 μL, compared to 50.0 ± 8.9% and 87.1 ± 3.3% observed for non-transferred embryos (i.e. embryos that remained in the same drops) for 5 and 20 μL, respectively. (p-value = 0.005 for 5 μL, and 0.03 for 20 μL in unpaired t-tests.) Transferred and non-transferred embryos developed to blastocysts at comparable rates of 94.0 ± 4.0% and 97.0 ± 2.1%, respectively, for embryos cultured in groups of ten.