Automated technology for the preservation of Drosophila embryos - PROJECT SUMMARY Drosophila research labs routinely maintain hundreds to thousands of unique strains. Not only is this practice labor and resource intensive, but there is a risk that valuable strains will be lost, contaminated, or acquire additional mutations. This limitation would be overcome by the implementation of Drosophila cryopreservation methods. Cryopreservation of Drosophila embryos was first reported nearly 30 years ago, with subsequent optimization that enables preservation of both wild type and transgenic lines. Broad implementation of these cryopreservation technologies has not yet been realized. This may be due in part to complex methodologies utilizing toxic solvents and desiccation steps required to permeabilize the embryos to cryoprotective agents (CPAs). Recently, our lab has developed an embryo permeabilization solution that is free of harsh solvents, does not require desiccation or manual mixing, and has been semi-automated using syringe pumps. We have demonstrated in preliminary data that this approach successfully enables cryopreservation of Drosophila embryos that develop into adults and produce offspring. In the present technology driven proposal, our overall goal is now to develop a fully autonomous fluidic device capable of performing all liquid handling steps (i.e. dechorionation, permeabilization and CPA loading) required in the cryopreservation protocol. Here, no external computers or mechanical systems are required, as the device automatically times and performs liquid handling using only power from house vacuum lines. This is significant, as it eliminates high upfront costs associated with the fluidic system, reduces user-error, requires less labor and resources, and can be multiplexed. We anticipate this approach will result in an accessible, affordable technology that can be broadly implemented by the research community regardless of laboratory size or operating budget. Key deliverables for this focused technology award include i) a robust and simple chemical process that enables permeabilization and CPA uptake in Drosophila embryos and ii) an autonomous fluidic device that enables precise timing and delivery of reagents. The scientific team is well-suited to perform the work described in this proposal and includes experts in chemistry and cryobiology (Sandlin), Drosophila biology (White) and engineering (Toner). Our work will be completed through three specific aims. In Aim 1, we will focus on optimization of the chemical process for the cryopreservation procedure (e.g. permeabilization, CPA loading, etc.). In Aim 2, we will fabricate and validate the fluidic device to achieve full automation of complex liquid handling steps. In Aim 3 we will complete a validation study and disseminate the technology. Specifically, we will integrate best practices from Aims 1/2 to validate the fully automated procedure against a larger panel of Drosophila strains. Our goal is to maintain ≥60% survival of thawed embryos to adults relative to non-preserved controls. We will further work with three outside Drosophila labs to identify and address variables that may impact full use and broad implementation of this technology.
