title: Microwells support high-resolution time-lapse imaging and development of preimplanted mouse embryos.
abstract: A vital aspect affecting the success rate of in vitro fertilization is the culture environment of the embryo. However, what is not yet comprehensively understood is the affect the biochemical, physical, and genetic requirements have over the dynamic development of human or mouse preimplantation embryos. The conventional microdrop technique often cultures embryos in groups, which limits the investigation of the microenvironment of embryos. We report an open microwell platform, which enables micropipette manipulation and culture of embryos in defined sub-microliter volumes without valves. The fluidic environment of each microwell is secluded from others by layering oil on top, allowing for non-invasive, high-resolution time-lapse microscopy, and data collection from each individual embryo without confounding factors. We have successfully cultured mouse embryos from the two-cell stage to completely hatched blastocysts inside microwells with an 89% success rate (n = 64), which is comparable to the success rate of the contemporary practice. Development timings of mouse embryos that developed into blastocysts are statistically different to those of embryos that failed to form blastocysts (p-value < 10(-10), two-tailed Student's t-test) and are robust indicators of the competence of the embryo to form a blastocyst in vitro with 94% sensitivity and 100% specificity. Embryos at the cleavage- or blastocyst-stage following the normal development timings were selected and transferred to the uteri of surrogate female mice. Fifteen of twenty-two (68%) blastocysts and four of ten (40%) embryos successfully developed into normal baby mice following embryo transfer. This microwell platform, which supports the development of preimplanted 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.
authors.affiliation: Institute of Nanoengineering and Microsystems, National Tsing Hua University , 30013 Hsinchu, Taiwan.
authors.forename: Yu-Hsiang
authors.initials: YH
authors.lastname: Chung
authors.affiliation: Institute of Nanoengineering and Microsystems, National Tsing Hua University , 30013 Hsinchu, Taiwan.
authors.forename: Yi-Hsing
authors.initials: YH
authors.lastname: Hsiao
authors.affiliation: Institute of Nanoengineering and Microsystems, National Tsing Hua University , 30013 Hsinchu, Taiwan.
authors.forename: Wei-Lun
authors.initials: WL
authors.lastname: Kao
authors.affiliation: Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes , 35053 Zhunan, Miaoli County, Taiwan.
authors.forename: Chia-Hsien
authors.initials: CH
authors.lastname: Hsu
authors.affiliation: Institute of Nanoengineering and Microsystems, National Tsing Hua University , 30013 Hsinchu, Taiwan.
authors.forename: Da-Jeng
authors.identifier: 0000-0001-6631-780X
authors.initials: DJ
authors.lastname: Yao
authors.affiliation: Institute of Nanoengineering and Microsystems, National Tsing Hua University , 30013 Hsinchu, Taiwan.
authors.forename: Chihchen
authors.initials: C
authors.lastname: Chen