3D Printed Organisms Enabled by Aspiration-Assisted Adaptive Strategies.

Devising an approach to deterministically position organisms can impact various fields such as bioimaging, cybernetics, cryopreservation, and organism-integrated devices. This requires continuously assessing the locations of randomly distributed organisms to collect and transfer them to target spaces without harm. Here, an aspiration-assisted adaptive printing system is developed that tracks, harvests, and relocates living and moving organisms on target spaces via a pick-and-place mechanism that continuously adapts to updated visual and spatial information about the organisms and target spaces. These adaptive printing strategies successfully positioned a single static organism, multiple organisms in droplets, and a single moving organism on target spaces. Their capabilities are exemplified by printing vitrification-ready organisms in cryoprotectant droplets, sorting live organisms from dead ones, positioning organisms on curved surfaces, organizing organism-powered displays, and integrating organisms with materials and devices in customizable shapes. These printing strategies can ultimately lead to autonomous biomanufacturing methods to evaluate and assemble organisms for a variety of single and multi-organism-based applications.

High-throughput genetic manipulation of multicellular organisms using a machine-vision guided embryonic microinjection robot.

Microinjection is a technique used for transgenesis, mutagenesis, cell labeling, cryopreservation, and in vitro fertilization in multiple single and multicellular organisms. Microinjection requires specialized skills and involves rate-limiting and labor-intensive preparatory steps. Here, we constructed a machine-vision guided generalized robot that fully automates the process of microinjection in fruit fly (Drosophila melanogaster) and zebrafish (Danio rerio) embryos. The robot uses machine learning models trained to detect embryos in images of agar plates and identify specific anatomical locations within each embryo in 3D space using dual view microscopes. The robot then serially performs a microinjection in each detected embryo. We constructed and used three such robots to automatically microinject tens of thousands of Drosophila and zebrafish embryos. We systematically optimized robotic microinjection for each species and performed routine transgenesis with proficiency comparable to highly skilled human practitioners while achieving up to 4× increases in microinjection throughput in Drosophila. The robot was utilized to microinject pools of over 20,000 uniquely barcoded plasmids into 1,713 embryos in 2 days to rapidly generate more than 400 unique transgenic Drosophila lines. This experiment enabled a novel measurement of the number of independent germline integration events per successfully injected embryo. Finally, we showed that robotic microinjection of cryoprotective agents in zebrafish embryos significantly improves vitrification rates and survival of cryopreserved embryos post-thaw as compared to manual microinjection. We anticipate that the robot can be used to carry out microinjection for genome-wide manipulation and cryopreservation at scale in a wide range of organisms.