Toward embryo cryopreservation-on-a-chip: A standalone microfluidic platform for gradual loading of cryoprotectants to minimize cryoinjuries

Tirgar, Pouria; Sarmadi, Fatemeh; Najafi, Mojgan; Kazemi, Parinaz; Azizmohseni, Sina; Fayazi, Samaneh; Zandi, Ghazaleh; Ziaie, Nikta; Naseri, Aida Shoushtari Zadeh; Ehrlicher, Allen J.; Dashtizad, Mojtaba

doi:10.1063/5.0047185

Cited by 14 publications

(7 citation statements)

References 76 publications

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“…Procedural variation—adherence to timing and ability to trace the sample—affects cell viability following warming [ 29 ]. In summary, vitrification is a manual and labor-intensive procedure that is technically challenging [ 13 , 30 , 31 ]. Thus, we hypothesized that vitrification and warming would be simplified with a procedure that reduces oocyte and embryo handling and improves traceability.…”

Section: Introductionmentioning

confidence: 99%

Vitrification within a nanoliter volume: oocyte and embryo cryopreservation within a 3D photopolymerized device

Yagoub

Lim

Tan

et al. 2022

J Assist Reprod Genet

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Purpose Vitrification permits long-term banking of oocytes and embryos. It is a technically challenging procedure requiring direct handling and movement of cells between potentially cytotoxic cryoprotectant solutions. Variation in adherence to timing, and ability to trace cells during the procedure, affects survival post-warming. We hypothesized that minimizing direct handling will simplify the procedure and improve traceability. To address this, we present a novel photopolymerized device that houses the sample during vitrification. Methods The fabricated device consisted of two components: the Pod and Garage. Single mouse oocytes or embryos were housed in a Pod, with multiple Pods docked into a Garage. The suitability of the device for cryogenic application was assessed by repeated vitrification and warming cycles. Oocytes or early blastocyst-stage embryos were vitrified either using standard practice or within Pods and a Garage and compared to non-vitrified control groups. Post-warming, we assessed survival rate, oocyte developmental potential (fertilization and subsequent development) and metabolism (autofluorescence). Results Vitrification within the device occurred within ~ 3 nL of cryoprotectant: this volume being ~ 1000-fold lower than standard vitrification. Compared to standard practice, vitrification and warming within our device showed no differences in viability, developmental competency, or metabolism for oocytes and embryos. The device housed the sample during processing, which improved traceability and minimized handling. Interestingly, vitrification-warming itself, altered oocyte and embryo metabolism. Conclusion The Pod and Garage system minimized the volume of cryoprotectant at vitrification—by ~ 1000-fold—improved traceability and reduced direct handling of the sample. This is a major step in simplifying the procedure.

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Section: Introductionmentioning

confidence: 99%

Vitrification within a nanoliter volume: oocyte and embryo cryopreservation within a 3D photopolymerized device

Yagoub

Lim

Tan

et al. 2022

J Assist Reprod Genet

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“…The presence of non-permeable CPAs lowers the chemical potential of water via osmosis. The suction decreases with increasing osmotic term, which lowers the stress placed on the membrane [ 121 ]. Furthermore, high concentrations of non-membrane-permeating CPAs present in lipid membranes decrease the occurrence of two dehydration damaging effects; that is, they decrease the occurrence of non-lamellar phases, and they lower the transition temperature [ 18 ].…”

Section: Applicable Cryoprotectants In Liposomal Freezingmentioning

confidence: 99%

The Role of Cryoprotective Agents in Liposome Stabilization and Preservation

Boafo

Magar

Ekpo

et al. 2022

IJMS

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To improve liposomes’ usage as drug delivery vehicles, cryoprotectants can be utilized to prevent constituent leakage and liposome instability. Cryoprotective agents (CPAs) or cryoprotectants can protect liposomes from the mechanical stress of ice by vitrifying at a specific temperature, which forms a glassy matrix. The majority of studies on cryoprotectants demonstrate that as the concentration of the cryoprotectant is increased, the liposomal stability improves, resulting in decreased aggregation. The effectiveness of CPAs in maintaining liposome stability in the aqueous state essentially depends on a complex interaction between protectants and bilayer composition. Furthermore, different types of CPAs have distinct effective mechanisms of action; therefore, the combination of several cryoprotectants may be beneficial and novel attributed to the synergistic actions of the CPAs. In this review, we discuss the use of liposomes as drug delivery vehicles, phospholipid–CPA interactions, their thermotropic behavior during freezing, types of CPA and their mechanism for preventing leakage of drugs from liposomes.

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“…Through precisely manipulating the fluids at the microscale, microfluidic systems can realize complex fluid behaviors such as co-flow diffusion, droplet generation, and concentration gradient generation using different channel designs [ 20 , 21 , 22 , 23 , 24 ]. For cryopreservation of oocytes and embryos [ 6 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ], two categories of microfluidic platforms—confinement devices and free-flow devices—have been developed to simplify the process of CPAs loading and unloading [ 33 ].…”

Section: Introductionmentioning

confidence: 99%

“…Confinement devices use various mechanisms (e.g., physical obstruction, pressure difference or electrowetting) to manipulate oocytes/embryos [ 6 , 25 , 26 , 27 , 28 , 29 , 30 , 32 ] and provide a temporal concentration gradient of CPAs around the cells to complete vitrification pretreatment. Guo et al designed a curved-channel and microcolumn array microfluidic device to immobilize the oocyte and linearly load CPAs under diffusion, which reduced the permeability damage caused by sudden changes in CPAs concentration [ 6 ].…”

Section: Introductionmentioning

confidence: 99%

“…Guo et al designed a curved-channel and microcolumn array microfluidic device to immobilize the oocyte and linearly load CPAs under diffusion, which reduced the permeability damage caused by sudden changes in CPAs concentration [ 6 ]. Tirgar et al fabricated a microchannel with a width smaller than the embryo size to limit the movement of the embryo, and integrated a capillary pump to introduce the CPAs instead of the microsyringe pump, which simplified the operation process [ 28 ]. Pyne et al developed the EWOD platform and used the digital microfluidic method to complete the vitrification pretreatment of embryos [ 26 ].…”

Section: Introductionmentioning

confidence: 99%

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Development of an Open Microfluidic Platform for Oocyte One-Stop Vitrification with Cryotop Method

et al. 2022

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Oocyte vitrification technology is widely used for assisted reproduction and fertility preservation, which requires precise washing sequences and timings of cryoprotectant agents (CPAs) treatment to relieve the osmotic shock to cells. The gold standard Cryotop method is extensively used in oocyte vitrification and is currently the most commonly used method in reproductive centers. However, the Cryotop method requires precise and complex manual manipulation by an embryologist, whose proficiency directly determines the effect of vitrification. Therefore, in this study, an automatic microfluidic system consisting of a novel open microfluidic chip and a set of automatic devices was established as a standardized operating protocol to facilitate the conventional manual Cryotop method and minimize the osmotic shock applied to the oocyte. The proposed open microfluidic system could smoothly change the CPA concentration around the oocyte during vitrification pretreatment, and transferred the treated oocyte to the Cryotop with a tiny droplet. The system better conformed to the operating habits of embryologists, whereas the integration of commercialized Cryotop facilitates the subsequent freezing and thawing processes. With standardized operating procedures, our system provides consistent treatment effects for each operation, leading to comparable survival rate, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) level of oocytes to the manual Cryotop operations. The vitrification platform is the first reported microfluidic system integrating the function of cells transfer from the processing chip, which avoids the risk of cell loss or damage in a manual operation and ensures the sufficient cooling rate during liquid nitrogen (LN2) freezing. Our study demonstrates significant potential of the automatic microfluidic approach to serve as a facile and universal solution for the vitrification of various precious cells.

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Toward embryo cryopreservation-on-a-chip: A standalone microfluidic platform for gradual loading of cryoprotectants to minimize cryoinjuries

Cited by 14 publications

References 76 publications

Vitrification within a nanoliter volume: oocyte and embryo cryopreservation within a 3D photopolymerized device

Vitrification within a nanoliter volume: oocyte and embryo cryopreservation within a 3D photopolymerized device

The Role of Cryoprotective Agents in Liposome Stabilization and Preservation

Development of an Open Microfluidic Platform for Oocyte One-Stop Vitrification with Cryotop Method

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