US-12616191-B2 - Systems and methods for reversible cryopreservation

US12616191B2US 12616191 B2US12616191 B2US 12616191B2US-12616191-B2

Abstract

Provided herein are systems, methods, and cryoprotective solutions for reversible cryopreservation of biological specimens, whole organs, and whole organisms. Exemplary methods include loading a cryoprotective agent into the biological specimen, cooling the biological specimen to a cryogenic temperature for preservation, storing the biological specimen at a preservation temperature state to preserve the biological specimen, rewarming the biological specimen by increasing a temperature of the biological specimen above the preservation temperature state, and unloading the cryoprotective agent from the biological specimen. The cooling is performed at a first rate to reduce ice formation, substantially homogeneously to reduce propensity for cracking of the preserved biological specimen, and at a first pressure to prevent or reduce ice expansion within the preserved biological specimen. The rewarming is performed at a second rate to reduce ice formation, substantially homogeneously to reduce propensity for cracking, and at a second pressure to prevent or reduce ice expansion.

Inventors

Fynn S.V.F. COMERFORD
Noah I. DANIEL
Katherine L.M. BANEY
Benjamin D. FELLOWS
Andrew P. Ulvestad
Itziar RÍOS RUIZ
Vassilis A. ALEXOPOULOS
Anna N. PUSHKIN
Hunter Cole Davis OZAWA
Hannah Z. SLABODKIN
Justin M. Olshavsky
Dhruv K. SUMATHI
Inga ZHURAVLEVA
Isla D.B. WEBER
John E. Bailey, III
Chen Tian

Assignees

UNTIL LABS INC.

Dates

Publication Date: 20260505
Application Date: 20250604

Claims (20)

1 . A system for cryopreserving a biological tissue, the system comprising: a perfusive cooling chamber for holding the biological tissue; one or more pumps configured to perfuse a plurality of fluids and a plurality of magnetic nanoparticles into the perfusive cooling chamber and into the biological tissue, the plurality of fluids comprising: a cryoprotective fluid comprising one or more cryoprotective agents, a first inert fluid, wherein the first inert fluid has a higher viscosity than the cryoprotective fluid, and a second inert fluid, wherein: in a first configuration, the one or more pumps perfuse the cryoprotective fluid into the perfusive cooling chamber and into the biological tissue, in a second configuration, the one or more pumps perfuse the first inert fluid into the perfusive cooling chamber and into the biological tissue, in a third configuration, the one or more pumps perfuse the second inert fluid into the perfusive cooling chamber and into the biological tissue, and in a fourth configuration, the one or more pumps perfuse the second inert fluid and the plurality of magnetic nanoparticles into the perfusive cooling chamber and into the biological tissue.
2 . The system of claim 1 , wherein the cryoprotective fluid, the first inert fluid, and the second inert fluid are perfused into cannulized vasculature of the biological tissue.
3 . The system of claim 1 , wherein the first inert fluid is perfused into the biological tissue such that it displaces the cryoprotective fluid within the biological tissue.
4 . The system of claim 1 , wherein the first inert fluid is immiscible with the cryoprotective fluid.
5 . The system of claim 1 , wherein the second inert fluid is perfused into the biological tissue such that it replaces the first inert fluid within the biological tissue.
6 . The system of claim 1 , wherein the second inert fluid is miscible with the first inert fluid.
7 . The system of claim 1 , wherein the second inert fluid has a lower viscosity than the first inert fluid.
8 . The system of claim 1 , wherein at least one of the first inert fluid and the second inert fluid is a fluorous fluid.
9 . The system of claim 1 , wherein the one or more cryoprotective agents comprise trimethylamine-N-oxide.
10 . The system of claim 1 , wherein the one or more cryoprotective agents comprise antifreeze proteins.
11 . The system of claim 10 , wherein the one or more antifreeze proteins are produced intracellularly via transient transfection, gene editing, or viral infection.
12 . The system of claim 1 , wherein, in the fourth configuration, a temperature of the second inert fluid is controlled to cause the biological tissue to cool from a first temperature to a second temperature.
13 . The system of claim 12 , wherein the first temperature is between 10° C. and −20° C.
14 . The system of claim 12 , wherein the second temperature is between −42° C. and −196° C.
15 . The system of claim 12 , wherein the plurality of fluids further comprises a third inert fluid having a lower viscosity than the second inert fluid, and wherein, in a fifth configuration: the one or more pumps perfuse the third inert fluid and the plurality of magnetic nanoparticles into the perfusive cooling chamber and into the biological tissue, and a temperature of the third inert fluid is controlled to cause the biological tissue to cool from the second temperature to a third temperature.
16 . The system of claim 1 , wherein the plurality of magnetic nanoparticles comprise a fluorous surface coating.
17 . The system of claim 1 , wherein the plurality of magnetic nanoparticles comprise core shell nanoparticles, the core shell nanoparticles comprising a CoFe core and a Mn shell.
18 . The system of claim 1 , wherein each of the plurality of magnetic nanoparticles has a diameter greater than or equal to 20 nm.
19 . A method of cryopreserving a biological tissue, the method comprising: perfusing the biological tissue with a cryoprotective fluid comprising one or more cryoprotective agents; perfusing the biological tissue with a first inert fluid such that the first inert fluid displaces the cryoprotective fluid within vasculature of the biological tissue; perfusing the biological tissue with a second inert fluid; perfusing the biological tissue with the second inert fluid and a plurality of magnetic nanoparticles; and controlling a temperature of the second inert fluid while perfusing the biological tissue with the second inert fluid and the plurality of magnetic nanoparticles to cause the biological tissue to cool from a first temperature to a second temperature.
20 . The method of claim 19 , further comprising: perfusing the biological tissue with a third inert fluid and the plurality of magnetic nanoparticles; and controlling a temperature of the third inert fluid while perfusing the biological tissue with the third inert fluid and the plurality of magnetic nanoparticles to cause the biological tissue to cool from the second temperature to a third temperature.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of International Application No. PCT/US2025/031949 filed Jun. 2, 2025, which claims the benefit of priority to U.S. Provisional Application No. 63/654,824 filed May 31, 2024, the contents of which are incorporated herein by reference in their entireties for all purposes. BACKGROUND Cryopreservation involves cooling biological constructs to very low temperatures for long term preservation. One approach to cryopreservation is vitrification, where biological constructs are cooled to cryogenic temperatures without the formation of ice. Ice can damage biological constructs, especially multi-cellular biological constructs, therefore it is advantageous to avoid ice formation during cryopreservation. Cryopreservation is a critical technology for the long-term storage and preservation of biological materials. However, current methods often result in suboptimal outcomes. Accordingly, new approaches are needed to preserve multicellular tissue constructs to high levels of viability. High-viability reversible cryopreservation of tissue, especially human tissue, could unlock progress in many areas of biology research. Neuroscience research, for example, may primarily rely on animal models as it is extremely difficult to experiment on fresh, functional human brain tissue, which could be more representative than animal models. The use of human brain tissue for research, enabled by higher viability preservation of that tissue, could significantly accelerate the translation of new findings to clinical applications. Ovarian tissue can be cryopreserved clinically for prepubescent female patients facing a malignant disease (for example, cancer) whose treatment may compromise the fertility of the tissue. Higher viability of preservation of this tissue could improve clinical outcomes and restore fertility to more patients undergoing this therapy. The same may be done for prepubescent male patients facing a malignant disease, with testicular tissue being cryopreserved for later implantation. Viable cryopreservation of transplantable organs, difficult or impossible using existing preservation techniques, would allow for better donor organ matching to recipients, stockpiling of organs for patients who need them in the future, and a less expensive organ transplantation process. Reversible cryopreservation of whole mammals and whole organisms would allow for preservation of sick patients with no current treatment options, but with a cure or treatment for their disease reasonably expected to be available in the future. It may also enable long distance human space flight, with astronauts preserved during long transit times and restored when they have reached their destination. SUMMARY A method for performing reversible cryopreservation on a biological specimen is disclosed. The method can include loading a cryoprotective agent into the biological specimen. The method can include (a) loading one or more cryoprotective agents into the biological specimen. The method can include (b) cooling the biological specimen to a cryogenic temperature for preservation. The method can include (c) storing the biological specimen at a preservation temperature state to preserve the biological specimen. The method can include (d) rewarming the biological specimen by increasing a temperature of the biological specimen above the preservation temperature state. The method can include (e) unloading the one or more cryoprotective agents from the biological specimen. The cooling in (b) can be performed (i) at a first rate to reduce ice formation, (ii) substantially homogeneously to reduce propensity for cracking of the preserved biological specimen, and/or (iii) at a first pressure to prevent or reduce ice expansion within the preserved biological specimen. The rewarming in (d) can be performed (i) at a second rate to reduce ice formation, (ii) substantially homogeneously to reduce propensity for cracking, and/or (iii) at a second pressure to prevent or reduce ice expansion. A method for reversible cryopreservation of a biological specimen is disclosed. The method can include (a) perfusing the a biological specimen with a cryoprotective agent and nanoparticles. The method can include (b) cooling the a biological specimen perfused in (a) using rapid volumetric cooling. The method can include (c) storing the cooled perfused a biological specimen in (b). The method can include (d) rewarming the cooled perfused a biological specimen in (b) or (c) using volumetric warming configured to excite the nanoparticles. The method can include (e) unloading the cryoprotective agent from the rewarmed a biological specimen in (d). A system for cooling and rewarming a biological specimen is disclosed. The system can have a container. The container can have a port in communication with a reservoir for holding a cryoprotective agent. The container can house a biological specimen. The system can have a press