Isochoric Pressure Based Preservation of Ovarian Tissue

ABSTRACT This project combines and further develops our ice-free isochoric vitrification platform, multi-step/multi-thermic machine perfusion, and next generation non-toxic cryopreservation cocktails to find a viable solution to the large public health need for fertility preservation in children and young adults after different diseases and disease treatments. Such technologies are urgently needed by 650,000 children and young adult cancer survivors in US, to help preserve fertility and restore endocrine function post toxic treatments. The NIH has recognized this as a top priority, the fourth of NICHDs Fertility and Infertility Branch’s SBIR priority research areas being focused on “Novel techniques for preservation of gametes and whole ovary and testes.”Using carefully optimized protocols and cryostasis cocktails, our team and others have successfully cryopreserved multiple tissues in an ice-free vitreous “glassy” or “amorphous” state, allowing for indefinite storage. Unfortunately, these advances in ice-free preservation have mostly not been successfully scaled beyond small-tissue and small-volume cell suspensions due to the high cryoprotectant chemical concentrations, and rapid cooling/warming rates necessary for current vitrification methods. However, our broader group together with Dr. Rubinsky at UC-Berkeley, have developed a scalable and biocompatible isochoric (constant volume) cryopreservation paradigm for completely ice-free vitrification.Building on the success of the Phase I feasibility study, this project will deliver: (i) an improved preservation and banking of ovarian tissues strips via ice-free equilibrium isochoric vitrification (compared to current gold-standard slow- freezing), (ii) comprehensive protocol for banking whole human ovaries with functional validation in human to mouse xeno-transplants and validation in non-human primates with embryo development and eventually healthy offspring, and (iii) clinically relevant prototype systems of isochoric vitrification, capable of banking whole ovaries (testes, or other similar size tissues) for years until re-plantation. In addition, this project provides tools, equipment, solutions and protocols with highly-translatable technology toward banking of whole vital organs, and other vascular grafts, addressing widespread needs for breakthroughs in biopreservation – from improved biospecimen preservation to vital organ banking and transplantation.To meet these objectives, in four specific aims, we (i) with the help of thermodynamic profiling, further optimize the composition of cryostasis solutions for isochoric vitrification with minimal pressures, (ii) refine the biocompatibility of the cocktails and protocols using ovarian tissue strips, with human-to-mouse xenotransplants and human blood vessels contractile functional assessment, (iii) scale to whole human ovary vitrification with multi-thermic machine perfusion and clinical grade isochoric devices, and as a capstone, (iv) cryobank whole non-human primate ovaries with ovarian tissue strips in vivo quality evaluation through minimally invasive autotransplantation, embryo development and eventual healthy offspring. Success of these novel approaches, individually or in combination, can enable further breakthroughs in oncofertility, biopreservation research, and clinical practice.