Pre-implantation Development of Mouse Embryos in Microwells

• Main Authors: Chung, Yu Hsiang, 鍾宇翔
• Other Authors: Chen, Chihchen
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/60023721727148669871

碩士 === 國立清華大學 === 奈米工程與微系統研究所 === 103 === Assisted reproductive technologies (ART) are of increasing importance and impact worldwide. A crucial aspect of ART is to control and mimic the in vitro environment of mammalian to the in vivo one. Integration biotechnology with micro- and nanotechnology is one of the necessary ways to improve ART. There are many platforms capable of nurturing cells in 3D, including using gel, microwells or hanging drop techniques. Microwells were chosen for the culture and investigation of early mouse embryos in this study, since microwells made of polydimethylsiloxane (PDMS) are low-cost, easy to operate, and biocompatible. Each mouse early embryo could be maintained in an individual microwell, allowing for high-resolution time-lapse microscopy and collecting the data of developmental process from every single embryo without confounding factors. In addition, the fluidic environment of each microwell could be secluded from each other by layering oil on top, preventing the communication of soluble factors between embryos cultured in individual microwells. The initial step of ART involves in vitro fertilization (IVF), and we demonstrated a successful fertilization rate of 67.9% in this research. We successfully cultured mouse embryos from the two-cell stage to blastocyst stage inside different volume of microwells with a ~80% successful rate. Among the five different volumes of microwells evaluated, we chose 393 nL microwells as the cultured platform to tracing the development process of mouse embryos. After cultured in microwells for 72 hours, 22 embryos at blastocyst stage were transferred into a recipient female mouse, and 15 mice were successfully born after 19 days. The development timings of mouse embryos that developed into blastocysts were statistically different to those of embryos that failed to form blastocysts (p–value < 10-10), and could be robust indicators of the quality of the embryo with >93% sensitivity and 100% specificity. This microwell platform, which supports the development of pre-implant embryos and is low-cost, easy to fabricate and operate, we believe, opens opportunities for a wide range of applications in reproductive medicine and cell biology.