Conduction‐Dominated Cryomesh for Organism Vitrification

Guo, Zongqi; Zuchowicz, Nikolas; Bouwmeester, Jessica; Joshi, Amey S.; Neisch, Amanda L.; Smith, Kieran; Daly, Jonathan; Etheridge, Michael L.; Finger, Erik B.; Kodandaramaiah, Suhasa B.; Hays, Thomas S.; Hagedorn, Mary; Bischof, John C.

doi:10.1002/advs.202303317

Cited by 4 publications

(2 citation statements)

References 69 publications

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“…While there exists an upper bound for the volume of CPA microinjectant that can be introduced into each embryo (10 nL), alternate strategies, for instance, robotic microdialysis of the yolk followed by microinjection of the equivalent of higher replacement CPA solution can be explored ( Jardine and Litvak 2003 ; Khosla et al . 2019 ; Guo et al . 2024 ).…”

Section: Discussionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

“…Microinjection can also be used to prepare model organisms such as zebrafish for cryopreservation ( Khosla et al . 2017 , 2020 ; Guo et al . 2024 ) or possibly Drosophila or other invertebrates with large relatively impermeable embryos in the future ( Zhan et al 2021 ).…”

Section: Introductionmentioning

confidence: 99%

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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Development of Macromolecular Cryoprotectants for Cryopreservation of Cells

Yuan,

Chen,

Zhu

et al. 2024

Macromol. Rapid Commun.

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Cryopreservation is a common way for long‐term storage of therapeutical proteins, erythrocytes, and mammalian cells. For cryoprotection of these biosamples to keep their structural integrity and biological activities, it is essential to incorporate highly efficient cryoprotectants. Currently, permeable small molecular cryoprotectants such as glycerol and dimethyl sulfoxide dominate in cryostorage applications, but they are harmful to cells and human health. As acting in the extracellular space, membrane‐impermeable macromolecular cryoprotectants, which exert remarkable membrane stabilization against cryo‐injury and are easily removed post‐thaw, are promising candidates with biocompatibility and feasibility. Water‐soluble hydroxyl‐containing polymers such as poly(vinyl alcohol) and polyol‐based polymers are potent ice recrystallization inhibitors, while polyampholytes, polyzwitterions, and bio‐inspired (glyco)polypeptides can significantly increase post‐thaw recovery with reduced membrane damages. In this review, the synthetic macromolecular cryoprotectants are systematically summarized based on their synthesis routes, practical utilities, and cryoprotective mechanisms. It provides a valuable insight in development of highly efficient macromolecular cryoprotectants with valid ice recrystallization inhibition activity for highly efficient and safe cryopreservation of cells.

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