SBIR-STTR Award

This program aims to develop a novel storage method for human ovarian tissue and whole ovaries, which can also be applied to testicular tissue and gonadal preservation as well as a variety of cells, tissues, and whole organs. This multi-pronged approach builds on recent advances in machine perfusion, nature-inspired cytoprotection strategies, and non-toxic cryoprotectant solutions, combining them with our novel method for constant-volume, pressure- assisted cooling. Our method promises to achieve vitrification of living tissues without the aid of toxic concentrations of cryoprotectants, in a system at thermodynamic equilibrium and potentially at temperatures as high as -70C. It ameliorates or entirely circumvents many of the limitations of conventional cryopreservation methods for large tissues, including damaging ice crystallization, deleterious changes in solute concentrations, and volumetric changes. This approach has the potential to enable indefinite banking of ovarian tissues, whole ovaries, and other living materials while dramatically limiting tissue injury currently associated with ex-vivo storage. The technical objective of this Phase 1 proposal is to develop an optimized cocktail and protocol for preservation and indefinite banking of ovarian tissue strips. We will develop our method across three specific aims. In Aim 1, will use well established methods developed in one of our labs for comprehensive thermodynamic characterization of novel, nature-inspired cryoprotectant solutions in an isochoric (constant-volume) system; this will allow us to select solutions that allow for pressure-assisted vitrification of living materials at low to intermediate pressures and much higher temperatures than traditionally needed for ice-free cryopreservation. In Aim 2, we select the most effective cryoprotectant solutions established in Aim 1 to optimize our "high sub-zero vitrification" in a cell-based model for ovarian tissue consisting of human primary ovarian epithelial cells, human primary ovarian granulosa cells, and human endothelial cells; using high-throughput combinatorial testing, we will select protocols which maximize cell viability and improve on current preservation standards. In Aim 3, we will test our optimized protocols for the preservation of human ovarian cortical tissue strips, along with one of the most successful protocols for ovarian tissue freezing and for ovarian tissue vitrification, respectively; we will additionally test the augmentation of these methods with subnormothermic machine perfusion, enabling further reduction of ischemic tissue injury. Success of these novel approaches individually or in combination will likely enable breakthroughs in clinical oncofertility and biopreservation.

Public Health Relevance Statement:
Cancer treatment and research is inhibited by limitations in storage of biomaterials of every scale, from whole ovaries, testes, and ovarian and testicular tissue to preserve the fertility of pediatric and young adult cancer patients, to the banking of patient biospecimens for research; it is no exaggeration that preservation breakthroughs can potentially save or change the lives of millions of cancer patients. We have developed a novel approach to dramatically increase the effectiveness of ovarian tissue banking, helping protect fertility and endocrine function in female cancer survivors. This approach should be widely applicable for preservation of living systems.

Project Terms:
Affect; Animals; biological material; Biomaterials; Biocompatible Materials; bioengineering; Biomedical Engineering; neoplasm/cancer; malignancy; Malignant Tumor; Cancers; Malignant Neoplasms; Cell Viability; Cell Survival; Cell Body; Cells; youngster; childrens'; children; Children (0-21); Child Youth; 0-11 years old; Child; life change; Climacteric; low temperature; cold temperature; cold storage; cold preservation; Cryofixation; Cryopreservation; Crystallization; Dromisol; Domoso; Dimethylsulphoxide; Dimethylsulphinyl; Demeso; Demasorb; DMSO; Dimethyl Sulfoxide; Disorder; Disease; Environment; Epithelial Cells; balance function; balance; Equilibrium; Female; Fecundity; Fecundability; Fertility; Freezing; Au element; Gold; Modern Man; Human; Ice; unable to bear children; infertile; fertility loss; fertility cessation; Difficulty conceiving; Cannot achieve a pregnancy; Infertility; heavy metal lead; heavy metal Pb; Pb element; Lead; Methods; Molecular Weight; Organ Procurements; living system; Organism; Ovary; Patients; Perfusion; Phosphatides; Phospholipids; preservation; Biologic Preservation; Biological Preservation; pressure; Primates Mammals; Primates; Public Health; Research; Safety; Temperature; Testing; Testicles; Testis; Thermodynamic; Thermodynamics; Tissue Collection; Tissue Banks; Body Tissues; Tissues; transplant; Transplantation; Woman; Premature Ovarian Failure; premature ovarian aging; Tissue Banking; Injury; base; Organ; improved; Ovarian; Clinical; Phase; Biological; Series; Survivors; Epithelial; Endothelial Cells; pediatric; Childhood; young adulthood; adult youth; young adult; Staging; Nature; Life; animation; programs; human tissue; Protocol; Protocols documentation; cell type; System; Endocrine; Cell Protection; Cytoprotection; high temperature; High temperature of physical object; Cell Volumes; experience; glucose analog; solute; success; novel; resection; Surgical Removal; Removal; Extirpation; Abscission; Excision; Modeling; anticancer therapy; Malignant Neoplasm Treatment; Malignant Neoplasm Therapy; Cancer Treatment; cancer therapy; Ovarian Granulosa Cell; Effectiveness; Testicular Parenchyma; Testicular Tissue; Cancer Patient; survive cancer; Cancer Survivor; clinical test; Clinical Testing; Clinical Evaluation; research clinical testing; Ovarian Tissue; Characteristics; developmental; Development; oncogenomics; cancer genomics; cancer research; anticancer research; conditioning; novel strategy; novel approaches; new approaches; novel strategies; efficacy trial; Resistance; resistant; combinatorial; Cell model; Cellular model; implantation; biobank; biorepository; young woman; adolescent women; adolescent woman; rate of change; genomic biomarker; genomic marker; reproductive organ; oncofertility

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.

Public Health Relevance Statement:
NARRATIVE Technologies for cryobanking of ovarian, and testicular, tissues are urgently needed to protect the fertility of, often children, patients undergoing treatment for cancer, blood diseases such as sickle cell, autoimmune and metabolic disorders, immune deficiencies, and other diseases that eventually lead to gonadal failure. As the survivorship rates for cancer increase (85% 5-year survival rate in childhood cancers) and fertility preservation recently being recognized by the American Society of Clinical Oncology and the American Society of Reproductive Medicine as one of the most prominent survivorship issues for these patients, we have developed a novel approach to indefinitely bank functional ovaries, to protect fertility and restore endocrine function of these patients post toxic treatments. Our comprehensive approach, using our ice-free isochoric vitrification and machine perfusion platforms, (i) brings a totally new capabliity to the fields of prepuberty girl and boy and young woman infertility and oncofertility, that (ii) also can provide life-changing solutions for service members, and (iii) eventually can be horizontally translated to other complex tissue and vital organ preservation, with (iv) transformative effect in many areas of medicine, including transplantation, tissue engineering, trauma care and biomedical research.

Project Terms:
Sickle Cell Anemia; Hb SS disease; HbSS disease; Hemoglobin S Disease; Hemoglobin sickle cell disease; Hemoglobin sickle cell disorder; sickle cell disease; sickle cell disorder; sickle disease; sicklemia; Autoimmune Diseases; autoimmune condition; autoimmune disorder; Biomedical Research; Blood Vessels; vascular; Bone Marrow; Bone Marrow Reticuloendothelial System; Malignant Neoplasms; Cancers; Malignant Tumor; malignancy; neoplasm/cancer; Child; 0-11 years old; Child Youth; Children (0-21); youngster; Climacteric; life change; Complement; Complement Proteins; Cryopreservation; Cryofixation; cold preservation; cold storage; Disease; Disorder; Dry Ice; Carbon Dioxide Snow; Embryonic Development; Embryo Development; Embryogenesis; Engineering; Equilibrium; balance; balance function; Equipment; Feasibility Studies; Fertility; Fecundability; Fecundity; Fishes; Food; Food or Food Product; Foundations; Freedom; Liberty; Freezing; Germ Cells; Gametes; Germ-Line Cells; Reproductive Cells; Sex Cell; initial cell; sexual cell; Gold; Growth; Generalized Growth; Tissue Growth; ontogeny; Hematological Disease; Blood Diseases; Hematologic Diseases; Hematological Disorder; blood disorder; Hormones; Endocrine Gland Secretion; Therapeutic Hormone; Human; Modern Man; Ice; Infertility; Cannot achieve a pregnancy; Difficulty conceiving; fertility cessation; fertility loss; infertile; Lead; Pb element; heavy metal Pb; heavy metal lead; Medicine; Metabolic Diseases; Metabolic Disorder; Thesaurismosis; metabolism disorder; Methods; Mus; Mice; Mice Mammals; Murine; United States National Institutes of Health; NIH; National Institutes of Health; Oocytes; Ovocytes; Organ Donor; Organ Preservation; Ovary; Patients; Perfusion; Periodicity; Cyclicity; Rhythmicity; pressure; Primates; Primates Mammals; Production; Protective Agents; Protective Drugs; Public Health; Rattus; Common Rat Strains; Rat; Rats Mammals; Surgical Replantation; Reimplantation; Replantation; Research; Science; Societies; sperm cell; Sperm; Spermatozoa; zoosperm; Continuance of life; Survivorship Issues; Survival Rate; survivorship; Suspensions; Suspension substance; Family suidae; Pigs; Suidae; Swine; porcine; suid; Technology; Temperature; Testis; Testicles; Thermodynamics; Thermodynamic; Tissue Preservation; Tissues; Body Tissues; Translating; Transplantation; transplant; Autologous Transplantation; Autograft; Autotransplant; autologous graft; autotransplantation; Competence; base; Organ; improved; Ovarian; Area; Clinical; Phase; Chemicals; Evaluation; Sickle Cell; sickle RBC; sickle erythrocyte; sickle red blood cell; Failure; Childhood; pediatric; prepuberty; Prepuberal state; prepubertal; Individual; nonhuman primate; non-human primate; Recovery; young adult; adult youth; young adulthood; Progress Reports; Reproductive Health; Reproductive Medicine; Malignant Childhood Neoplasm; Childhood Cancers; Malignant Childhood Tumor; Malignant Pediatric Neoplasm; Malignant Pediatric Tumor; Malignant childhood cancer; cancer in a child; cancer in children; child with cancer; childhood malignancy; children with cancer; pediatric cancer; pediatric malignancy; Letters; tool; Research Priority; Immunes; Immune; Complex; Autologous; Clinic; Protocol; Protocols documentation; System; restoration; Heterograft; Heterologous Transplantation; Xenograft; Xenotransplantation; xeno-transplant; xeno-transplantation; Xenograft procedure; Endocrine; high temperature; High temperature of physical object; American; bioengineered tissue; engineered tissue; Tissue Engineering; biocompatibility; biomaterial compatibility; Cell Volumes; experience; success; cryogenics; Stretching; novel; offspring; Devices; Vascular Graft; Modeling; boys; girls; preconditioning; Cancer Treatment; Malignant Neoplasm Therapy; Malignant Neoplasm Treatment; anti-cancer therapy; anticancer therapy; cancer-directed therapy; cancer therapy; Address; Cryopreserved Tissue; NICHD; National Institute of Children's Health and Human Development; National Institute of Child Health and Human Development; Preservation Technique; Reproductive Physiology; Testicular Tissue; Testicular Parenchyma; in vivo; American Society of Clinical Oncology; ASCO; Cancer Survivor; survive cancer; Ovarian Tissue; Small Business Innovation Research Grant; SBIR; Small Business Innovation Research; Validation; Development; developmental; design; designing; next generation; novel strategies; new approaches; novel approaches; novel strategy; scale up; innovation; innovate; innovative; combinatorial; clinically relevant; clinical relevance; clinical application; clinical applicability; prototype; commercialization; minimally invasive; clinical practice; phase 2 study; phase II study; young woman; adolescent woman; adolescent women; trauma care; pediatric patients; child patients; Formulation; reproductive organ; oncofertility; service member; military member; preservation; fertility preservation; preserve fertility; Chemotherapy and/or radiation; Chemotherapy and Radiation