Compositions and methods to protect mammalian tissue against cold and other metabolic stresses
Inventors
Ou, Jingxing • Li, Wei • Miyagishima, Kiyoharu Josh
Assignees
Usa REPRESENTED BY SECRETARY Dhhs AS • US Department of Health and Human Services
Publication Number
US-11547708-B2
Publication Date
2023-01-10
Expiration Date
2038-08-20
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Abstract
A composition for preserving viability of cells, tissues, or organs at a low temperature is provided. The composition includes a mitochondrial uncoupling agent, at least one protease inhibitor, and a reducing agent. Methods to protect cells, tissues, or organs from exposure to cold and other metabolic stress are also provided.
Core Innovation
The invention provides a composition and methods for preserving the viability of mammalian cells, tissues, or organs during exposure to low temperature environments. The composition includes three key components: a mitochondrial uncoupling agent, at least one protease inhibitor, and a reducing agent. This composition aims to protect cells, tissues, or organs from cold and other metabolic stresses, enabling their survival and recovery following exposure to severe cold conditions.
The problem addressed by the invention arises from the limitations of therapeutic hypothermia methods and transplantation procedures. Mild and moderate hypothermia can protect brain injuries but are associated with complications such as fluid and electrolyte imbalances, arrhythmias, insulin resistance, shivering, and coagulation problems. Severe therapeutic hypothermia at around 4-5° C. is postulated to be more beneficial but currently induces irreversible damage and increased complications. Additionally, isolated neural cells or tissues are challenging to maintain viable at low temperatures prior to transplantation. Although antioxidants have been incorporated in standard cold storage solutions like University of Wisconsin (UW) solution to alleviate oxidative stress, cold-induced microtubule disruption still occurs. Therefore, there is a need for improved compositions and methods to better preserve cells, tissues, and organs at low temperatures, addressing both therapeutic hypothermia and transplantation challenges.
The invention discloses that targeting mitochondrial activities and protein degradation machinery using the novel composition surprisingly confers cold resistance to non-hibernating mammalian tissues, as tested in neuronal cells, retinal explants, and kidney tissues. Specifically, the mitochondrial uncoupling agent reduces mitochondrial hyperpolarization and reactive oxygen species (ROS) production caused by cold stress. Protease inhibitors prevent damage from leaked lysosomal proteases due to lysosomal membrane permeabilization. The reducing agent helps mitigate oxidative damage. The combined treatment preserves microtubule morphology, tubulin protein levels, and functional viability of cells and tissues after cold exposure, supporting their use during low-temperature storage, hypothermic therapies, transplantation, or induced hibernation.
Claims Coverage
The patent includes one independent claim focusing on a method for protecting mammalian cells, tissues, or organs from low-temperature injury using a composition with specific components.
Composition comprising a mitochondrial uncoupling agent
A compound represented by the chemical Formula (I) acting as a mitochondrial uncoupling agent to modulate mitochondrial membrane potential and reduce cold-induced reactive oxygen species.
Inclusion of specific protease inhibitors
At least one protease inhibitor including combinations of E-64, Pepstatin A, AEBSF, and Bestatin to prevent proteolytic damage caused by lysosomal membrane permeabilization during cold stress.
Use of a reducing agent
A reducing agent such as ascorbic acid included to mitigate oxidative damage occurring during cold exposure.
Combination with cryopreservation agents
The composition can be admixed with cryopreservation agents like University of Wisconsin (UW) solution to form a preservation solution for cells, tissues, or organs under hypothermic storage.
Applicable to a wide range of mammalian cells, tissues, or organs
The method applies to neuronal cells, retinal cells, corneal cells and tissue, skin, heart, lung, pancreas, kidney, liver, intestines, stem cells, blood, and other transplantable tissues.
Treatment parameters for cold storage
Temperature ranging from 2° C. to 5° C. and treatment durations from 4 to 72 hours are specified for preservation efficacy.
The independent claim describes a method to protect mammalian cells, tissues, or organs from injury at low temperatures by contacting them with a composition comprising a mitochondrial uncoupling agent of formula (I), specific protease inhibitors, and a reducing agent, optionally combined with cryopreservation agents. The method is applicable to various transplantable cells or organs and includes defined concentration ranges and treatment parameters to enhance preservation during hypothermic conditions.
Stated Advantages
The composition preserves microtubule morphology and tubulin protein levels in cells subjected to prolonged cold exposure.
It reduces mitochondrial hyperpolarization and oxidative stress that lead to cell damage under hypothermic conditions.
Protease inhibitors prevent degradation of cellular proteins by proteases leaked due to lysosomal membrane permeabilization.
The combined treatment improves functional viability and response of retinal ganglion cells after cold storage.
The composition enhances preservation of tissues and organs such as kidneys during cold storage and promotes recovery upon rewarming.
Documented Applications
Use in therapeutic hypothermia to protect brain injury or trauma and improve surgery outcomes.
Preservation of isolated neural cells or tissues prior to transplantation.
Storage of donor organs such as kidneys under cold ischemic conditions to prolong viability before transplantation.
Protection of retinal explants and neurons from cold-induced damage.
Potential induction of hibernation or protection against hypothermia in non-hibernating mammals.
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