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

Abstract

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.

Comparative study on in vitro fertilization, cleavage and blastocyst development of post-warmed vitrified cattle oocytes in different media

“…In this study, blastocyst development from vitrified oocytes was low in all treatments, showing that vitrification still reduces developmental competence even with improvements in fertilization and culture methods. This study contradicts with results found by Yagoub et al ( 34 ), who found higher cleavage and blastocyst survival in vitrified oocytes using Pods and the Garage system and cultured with VitroCleave PLUS ™ . The difference may be due to the methods used, as our study relied on a conventional straw-based protocol, while Yagoub et al ( 34 ) used a nanoliter device designed to improve cooling and warming efficiency.…”

“…This study contradicts with results found by Yagoub et al ( 34 ), who found higher cleavage and blastocyst survival in vitrified oocytes using Pods and the Garage system and cultured with VitroCleave PLUS ™ . The difference may be due to the methods used, as our study relied on a conventional straw-based protocol, while Yagoub et al ( 34 ) used a nanoliter device designed to improve cooling and warming efficiency. Improving outcomes will require an integrated approach: optimizing cryoprotectant selection, cooling/warming rates, fertilization medium, and culture conditions.…”

Comparative study on in vitro fertilization, cleavage and blastocyst development of post-warmed vitrified cattle oocytes in different media

“…In this study, blastocyst development from vitrified oocytes was low in all treatments, showing that vitrification still reduces developmental competence even with improvements in fertilization and culture methods. This study contradicts with results found by Yagoub et al ( 34 ), who found higher cleavage and blastocyst survival in vitrified oocytes using Pods and the Garage system and cultured with VitroCleave PLUS ™ . The difference may be due to the methods used, as our study relied on a conventional straw-based protocol, while Yagoub et al ( 34 ) used a nanoliter device designed to improve cooling and warming efficiency.…”

“…This study contradicts with results found by Yagoub et al ( 34 ), who found higher cleavage and blastocyst survival in vitrified oocytes using Pods and the Garage system and cultured with VitroCleave PLUS ™ . The difference may be due to the methods used, as our study relied on a conventional straw-based protocol, while Yagoub et al ( 34 ) used a nanoliter device designed to improve cooling and warming efficiency. Improving outcomes will require an integrated approach: optimizing cryoprotectant selection, cooling/warming rates, fertilization medium, and culture conditions.…”

Nano-liter perfusion microfluidic device made entirely by two-photon polymerization for dynamic cell culture with easy cell recovery

“…1 D). This basic structure can be observed in related previous studies, where the cradle is referred to as a ‘pod’ 40 , 41 . Two distinct features were added to the cradle in this study; the fin (denoted ‘g’ in Fig.…”

Systematic review and meta-analysis on patented and non-patented vitrification processes to ovarian tissue reported between 2000 and 2021

“…In animal species, this tool directly contributes to the implantation of germplasm banks, being fundamental to preserve the genetic biodiversity and the reproductive potential of rare and valuable animals (Santos et al, 2010;Comizzoli, 2017). In this regard, vitrification has been widely investigated as a method for ovarian cryopreservation, as it is widely used for the preservation of mammalian blastocysts and oocytes (Yagoub et al, 2022). In addition, it can reduce excessive ice crystal formation during the temperature-cooling process, minimizing the risk of mechanical damage to stromal and follicular cells (Leonel et al, 2019).…”

Comparative study on in vitro fertilization, cleavage and blastocyst development of post-warmed vitrified cattle oocytes in different media

“…In this study, blastocyst development from vitrified oocytes was low in all treatments, showing that vitrification still reduces developmental competence even with improvements in fertilization and culture methods. This study contradicts with results found by Yagoub et al ( 34 ), who found higher cleavage and blastocyst survival in vitrified oocytes using Pods and the Garage system and cultured with VitroCleave PLUS ™ . The difference may be due to the methods used, as our study relied on a conventional straw-based protocol, while Yagoub et al ( 34 ) used a nanoliter device designed to improve cooling and warming efficiency.…”

“…This study contradicts with results found by Yagoub et al ( 34 ), who found higher cleavage and blastocyst survival in vitrified oocytes using Pods and the Garage system and cultured with VitroCleave PLUS ™ . The difference may be due to the methods used, as our study relied on a conventional straw-based protocol, while Yagoub et al ( 34 ) used a nanoliter device designed to improve cooling and warming efficiency. Improving outcomes will require an integrated approach: optimizing cryoprotectant selection, cooling/warming rates, fertilization medium, and culture conditions.…”

Nano-liter perfusion microfluidic device made entirely by two-photon polymerization for dynamic cell culture with easy cell recovery

“…1 D). This basic structure can be observed in related previous studies, where the cradle is referred to as a ‘pod’ 40 , 41 . Two distinct features were added to the cradle in this study; the fin (denoted ‘g’ in Fig.…”

Systematic review and meta-analysis on patented and non-patented vitrification processes to ovarian tissue reported between 2000 and 2021

“…In animal species, this tool directly contributes to the implantation of germplasm banks, being fundamental to preserve the genetic biodiversity and the reproductive potential of rare and valuable animals (Santos et al, 2010;Comizzoli, 2017). In this regard, vitrification has been widely investigated as a method for ovarian cryopreservation, as it is widely used for the preservation of mammalian blastocysts and oocytes (Yagoub et al, 2022). In addition, it can reduce excessive ice crystal formation during the temperature-cooling process, minimizing the risk of mechanical damage to stromal and follicular cells (Leonel et al, 2019).…”

Comparative study on in vitro fertilization, cleavage and blastocyst development of post-warmed vitrified cattle oocytes in different media

“…In this study, blastocyst development from vitrified oocytes was low in all treatments, showing that vitrification still reduces developmental competence even with improvements in fertilization and culture methods. This study contradicts with results found by Yagoub et al ( 34 ), who found higher cleavage and blastocyst survival in vitrified oocytes using Pods and the Garage system and cultured with VitroCleave PLUS ™ . The difference may be due to the methods used, as our study relied on a conventional straw-based protocol, while Yagoub et al ( 34 ) used a nanoliter device designed to improve cooling and warming efficiency.…”

“…This study contradicts with results found by Yagoub et al ( 34 ), who found higher cleavage and blastocyst survival in vitrified oocytes using Pods and the Garage system and cultured with VitroCleave PLUS ™ . The difference may be due to the methods used, as our study relied on a conventional straw-based protocol, while Yagoub et al ( 34 ) used a nanoliter device designed to improve cooling and warming efficiency. Improving outcomes will require an integrated approach: optimizing cryoprotectant selection, cooling/warming rates, fertilization medium, and culture conditions.…”

Nano-liter perfusion microfluidic device made entirely by two-photon polymerization for dynamic cell culture with easy cell recovery

“…1 D). This basic structure can be observed in related previous studies, where the cradle is referred to as a ‘pod’ 40 , 41 . Two distinct features were added to the cradle in this study; the fin (denoted ‘g’ in Fig.…”

Systematic review and meta-analysis on patented and non-patented vitrification processes to ovarian tissue reported between 2000 and 2021

“…In animal species, this tool directly contributes to the implantation of germplasm banks, being fundamental to preserve the genetic biodiversity and the reproductive potential of rare and valuable animals (Santos et al, 2010;Comizzoli, 2017). In this regard, vitrification has been widely investigated as a method for ovarian cryopreservation, as it is widely used for the preservation of mammalian blastocysts and oocytes (Yagoub et al, 2022). In addition, it can reduce excessive ice crystal formation during the temperature-cooling process, minimizing the risk of mechanical damage to stromal and follicular cells (Leonel et al, 2019).…”