Oral Presentation ESA-SRB-APEG-NZSE 2022

Vitrification within a nanolitre volume: oocyte and embryo cryopreservation within a 3D photopolymerised device (#83)

Megan Lim 1 2 3 4 , Suliman Yagoub 1 2 4 , Cheow Yuen (Tiffany) Tan 1 2 4 , Darren Chow 1 2 4 , Kishan Dholakia 3 4 5 6 , Brant Gibson 1 7 , Jeremy Thompson 1 2 4 8 , Kylie Dunning 1 2 4
  1. Australian Research Council Centre for Nanoscale BioPhotonics, The University of Adelaide, Adelaide, South Australia, Australia
  2. Robinson Research Institute, School of Biomedicine, The University of Adelaide, Adelaide, South Australia, Australia
  3. The University of Adelaide, School of Biological Sciences, The University of Adelaide, Adelaide, SA, Australia
  4. Institute for Photonics and Advanced Sensing (IPAS), University of Adelaide, Adelaide, South Australia, 5000, Australia
  5. School of Physics and Astronomy, University of St Andrews, North Haugh, Scotland KY169SS
  6. Department of Physics, College of Science, Yonsei University, Seoul 03722, South Korea
  7. School of Science, Royal Melbourne Institute of Technology, Melbourne, Victoria, 3001, Australia
  8. Fertilis Pty Ltd, Adelaide, South Australia, 5005, Australia

Cryopreservation is an essential assisted reproductive technology widely practiced in the IVF clinic, providing the valuable opportunity for fertility preservation. The procedure is technically demanding, requiring precise handling of cells by an experienced embryologist within a strict timeframe to ensure minimal transfer of potentially cytotoxic cryoprotectants. The embryologist must meticulously trace cells throughout the process and avoid procedural deviations that affect cell survival post-warming, and subsequently fertilisation and embryo development. We hypothesised that minimising direct handling will simplify the procedure, improve traceability and consequently, cell viability. To address this, we present a novel 3D photopolymerised device that houses cells during vitrification and warming. The fabricated device consists of two components: the Pod (670 x 235 x 353 µm; l x w x h) and Garage (1150 x 450 x 345 µm). Individual mouse oocytes or embryos were housed in a Pod, with three Pods docked into a Garage. We assessed the suitability of the device for cryogenic application by examining its structural integrity following repeated vitrification and warming cycles. To understand how the device affected cell viability post-warming, we evaluated oocyte and embryo survival, developmental potential (fertilisation and subsequent embryo development) and metabolic profile by measuring endogenous fluorophores using confocal microscopy. Oocytes or blastocyst-stage embryos were vitrified either using standard practice or within Pods and a Garage and compared to non-vitrified control groups. Vitrification within the device occurred within ~3 nL of cryoprotectant: this volume being ~1000-fold lower than standard vitrification. We demonstrate that vitrification and warming within the device had comparable oocyte and embryo survival, developmental competency, and metabolic profile to that of standard practice. The Pod and Garage device minimised the volume of cytotoxic cryoprotectant at vitrification, improved traceability and reduced direct handling of the sample, paving the way for a major step in simplifying the procedure.