In vitro production efficiencies are low. The propose of this project is to develop and test a micro in vitro production (microIVP) device for the production of mammalian embryos. We believe that the integration of multiple IVP processes will provide further improvements in performance. The gains will be achieved via reduced handling and a reduction in variability as well as reduced stress manipulations. OBJECTIVES: We propose to develop and test a micro in vitro production (microIVP) device for the production of mammalian embryos. The microfluidic-based device will integrate maturation, fertilization, cumulus removal and culture. The ability to perform multiple IVP processes within a single device provides a basis for future automated, high throughput IVP systems that could decentralize animal production and allow unprecedented scientific research via enhanced functional capabilities. APPROACH: Once an oocyte(s) is placed into the device it is not removed until it is ready for transfer into a recipient animal. Because separate processes (maturation, culture, cumulus removal) when performed in microchannels have been shown to be beneficial, we believe that the integration of multiple IVP processes will provide further improvements in performance. The gains will be achieved via reduced handling and a reduction in variability as well as reduced stress manipulations (e.g. microfluidic cumulus removal). The microIVP device will be tested using bovine IVP. PROGRESS: 2004/05 TO 2005/12 The primary objective of the Phase I work was to design and construct a microfluidic device capable of supporting all stages of in vitro mammalian embryo production, from oocyte maturation through blastocyst formation The microfluidic-based device was designed to integrate maturation, fertilization, cumulus removal and culture. The basic idea was that, once an oocyte(s) was placed into the device it was not removed until it is ready for transfer into a recipient animal. The uIVP device was fabricated using elastomeric micromolding methods and tested using a bovine in vitro embryo production (IVP) scheme. Two basic categories of devices were constructed. In one category the inlet/culture channel dimensions were based on our dedicated cumulus removal device, which was 500um wide by 500um tall in the area nearest the suction ports. In the other category, the inlet/culture channels are 1000um wide by 275um tall, which is based on the culture channel of the dedicated embryo culture device. Several studies were completed including maturation and integrated IVP. In the maturation study, no significant differences were observed between oocyte maturation, as measured by blastocyst development, two different channel dimensions and between the VC design and the control treatment in the production of day 7 blastocysts. In the integrated device study, there were no significant differences in the number of blastocysts between most of the treatments. The limited number of COCs that were cultured in the microfluidic devices may have contributed to this result as well as the variance in average cell number of blastocysts across all treatments. While low numbers of embryos developed after complete production in microfluidic devices, some blastocysts still made it through complete production in microfluidic devices, including maturation, fertilization, cumulus removal and static culture. There are a number of factors at play throughout the integrated process, indicating that an effective system can be developed for complete production of IVP embryos in microfluidic devices. We have demonstrated equal or improved development to blastocyst for each individual microfluidic IVP step as compared to traditional methods (i.e. microdrops/Nunc well, vortexing). In addition, in our Phase I project we accomplished several important things. First, the integration of all IVP steps in a single devices supported blastocyst development demonstrating technical feasibility. Second, several issues unique to performing all IVP steps within a single device were discovered (e.g. plating, device operation issues). These two accomplishments provide us with the information needed to achieve our longer term goal of further improvements in development and pregnancy rates via the optimization of our integrated system. IMPACT: 2004/05 TO 2005/12 The microfluidic-based device integrates maturation, fertilization, cumulus removal and culture. Once an oocyte(s) is placed into the device it is not removed until it is ready for transfer into a recipient animal. Because separate processes (maturation, culture, cumulus removal, fertilization) when performed in microchannels have been shown to be beneficial, the integration and optimization of multiple IVP processes in a single microfluidic device should provide further improvements in performance. The ability to perform multiple IVP processes within a single device provides a basis for future automated, high throughput IVP systems that could centralize animal embryo production and allow unprecedented scientific research via enhanced functional capabilities
