Effects of culture medium and volume on preimplantation development of mouse embryos

Assisted reproductive technologies (ART) is gaining its importance worldwide, and a crucial aspect of ART is to control and mimic the in vitro environment. The first step of ART is in vitro fertilization, and we demonstrate the fertilization rate of ~60% (N = 37) when the oocytes and the sperms were cultured in human tubal fluid medium (HTF) first. In this study, we used microwells of different volume, ranging from 16 to 9,813 nL, as the platform to culture pre-implant mouse embryos. Microwells made of polydimethylsiloxane (PDMS) are low-cost, easy to operate, and biocompatible. Besides, the fluidic environment of each microwell could be secluded from each other by layering oil on top, allowing for data collection from each individual embryo without confounding factors. We successfully cultured mouse embryos from the two-cell stage to blastocyst stage inside different volume of microwells with a ~80% successful rate (N = 588), which is comparable to the successful rate of the conventional microdrop methods. Among the five different volumes of microwells evaluated, the blastocyst rater were higher when embryos were cultured in 3,533 nL and 393 nL microwells.To our surprise, microwells, as small as 200 μm in diameter and 16 nL in volume could still support the embryo to develop to blastocyst with a ~75% successful rate (N = 78).