Robotic platform for automated microinjection of zebrafish embryos for cryopreservation application

Joshi, Amey; Alegria, Andrew D; Smith, Kieran; Gohl, Daryl M.; Khosla, Kanav; Bischof, John C.; Kodandaramaiah, Suhasa B.

doi:10.1016/j.cryobiol.2022.11.143

Cited by 2 publications

(2 citation statements)

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“…First, high-concentration CPA (10 nL of 80 wt% PG + 20 wt% MeOH) was microinjected into the yolk of a zebrafish embryo at the high cell stage, 3.3 h after fertilization, [61] (0 min, Figure 6B) using robotic microinjection. In the previous protocol, [58,59,62] this microinjection step included gold nanorods for laser rewarming of the embryos. The gold nanorods were not included since the focus of this demonstration was on vitrification.…”

Section: Zebrafish Embryo Cryopreservationmentioning

confidence: 99%

“…The custom-built robotic microinjection system was a computer vision-guided robot that used off-the-shelf components to fully automate the microinjection procedure. [62][63][64] The CPA-injected embryos (1 min, Figure 6B) were transferred into an incubator at 28 °C to allow for CPA diffusion inside the yolk. After a 3-h recovery period, the embryos were placed on a cryomesh and immersed in a precooling bath (2.7 M PG + 1.2 M MeOH + 0.5 M trehalose) for 5 min.…”

Section: Zebrafish Embryo Cryopreservationmentioning

confidence: 99%

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Conduction‐Dominated Cryomesh for Organism Vitrification

Guo,

Zuchowicz,

Bouwmeester

et al. 2023

Advanced Science

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Vitrification‐based cryopreservation is a promising approach to achieving long‐term storage of biological systems for maintaining biodiversity, healthcare, and sustainable food production. Using the “cryomesh” system achieves rapid cooling and rewarming of biomaterials, but further improvement in cooling rates is needed to increase biosystem viability and the ability to cryopreserve new biosystems. Improved cooling rates and viability are possible by enabling conductive cooling through cryomesh. Conduction‐dominated cryomesh improves cooling rates from twofold to tenfold (i.e., 0.24 to 1.2 × 105 °C min−1) in a variety of biosystems. Higher thermal conductivity, smaller mesh wire diameter and pore size, and minimizing the nitrogen vapor barrier (e.g., vertical plunging in liquid nitrogen) are key parameters to achieving improved vitrification. Conduction‐dominated cryomesh successfully vitrifies coral larvae, Drosophila embryos, and zebrafish embryos with improved outcomes. Not only a theoretical foundation for improved vitrification in µm to mm biosystems but also the capability to scale up for biorepositories and/or agricultural, aquaculture, or scientific use are demonstrated.

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Section: Zebrafish Embryo Cryopreservationmentioning

confidence: 99%

Section: Zebrafish Embryo Cryopreservationmentioning

confidence: 99%

Conduction‐Dominated Cryomesh for Organism Vitrification

Guo,

Zuchowicz,

Bouwmeester

et al. 2023

Advanced Science

Self Cite

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

Alegria,

Joshi,

Mendana

et al. 2024

Genetics

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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 4x 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 two 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.

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Robotic platform for automated microinjection of zebrafish embryos for cryopreservation application

Cited by 2 publications

References 0 publications

Conduction‐Dominated Cryomesh for Organism Vitrification

Conduction‐Dominated Cryomesh for Organism Vitrification

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

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