ATP-Bio

TA1_001. Apoptosis inhibitors to reduce preservation injury

Korkut Uygun, MGH
Martin Yarmush, MGH
Basak Uygun, MGH
Mehmet Toner, MGH
Shannon Stott, MGH

Understand the differences in response to preservation injury and to various new CPAs and additives between different cell types

Ischemic injury during preservation leads to apoptosis (controlled cell death), which is acceptable and even required for healthy regeneration, but in large amounts can lead to graft failure after transplant. Our initial focus was on the effect of the already-approved pan-caspase inhibitor Emricasan on whole organs such as livers, to increase the viability after preservation. Our efforts have now transitioned to cells to understand the differences in response to preservation injury and to various new CPAs and additives between different cell types. Analyzing cell testbeds allows to tease apart differences between cell types and various high subzero preservation modalities in a cost efficient manner as well as identify novel CPAs on post-preservation viability.

TA1_002. Controlled, linear gradient delivery of permeable and non-permeable CPAs via hydrogel beads and viscoelastic poration

Mehmet Toner, MGH
Rebecca Sandlin, MGH

Deliver diverse CPAs to mammalian cells ex vivo in precisely timed multiple steps to avoid rapid fluctuations in cell volume

Intracellular loading of CPAs into target cells is a critical step for cryopreservation to avoid formation of lethal ice crystals. Biological membranes are usually much less permeable to CPAs than to water, resulting in high osmotic pressures and osmotic damage during the CPA loading and unloading phases. The main objective of this project is to efficiently deliver diverse CPAs to mammalian cells ex vivo in precisely timed multiple steps to avoid rapid fluctuations in cell volume. The methods are based on novel microfluidics and biomaterials to control biomolecule transport and plasma membrane permeability. One method uses hydrogel beads to slowly release CPA and equilibrate samples with minimized osmotic damage. Another method, viscoelastic poration consists of stretching the plasma membrane of individual cells until mechanical failure. Membrane integrity is restored within a few seconds. This very fast and robust process has the potential to efficiently deliver diverse CPAs and other large biomolecules to control the fate of ice inside cells during cryopreservation.

TA1_003. Non-invasive quantification of mitochondrial redox state using Resonance Raman Spectroscopy

Shannon Tessier, MGH
Korkut Uygun, MGH
Mehmet Toner, MGH
Paul Iaizzo, UMN
Timothy Pruett, UMN

Resonance Raman spectroscopy (RRS) to provide a rapid, non-invasive, and label-free diagnostic to assess the viability of cryopreserved cells

Cold ischemic injury is a critical problem during preservation of livers that can lead to life-threatening complications and substantially limit the utilization of livers for transplantation. However, because there are no early diagnostics available, fulminant injury may only become evident post-transplant. Mitochondria , and more particularly changes in the mitochondrial redox state play a central role in cold ischemic injury and are an ideal diagnostic target. In this study we used resonance Raman spectroscopy (RRS) to provide a rapid, non-invasive, and label-free diagnostic for quantification of the mitochondrial redox status. The data demonstrate the promise of monitoring dynamic changes in mitochondria as a function of perfusion. This novel diagnostic may be used in the future as a tool to better assess the viability of a range of cryopreserved cells, tissues, organs, and even whole organisms.

TA1_004. Partial freezing of hearts for transplantation

Shannon Tessier, MGH
Mehmet Toner, MGH
Martin Yarmush, MGH
Korkut Uygun, MGH

Using zebrafish model to test if apoptosis inhibitors can rescue cardiac function after experiencing ischemic injury 

The short time organs can be stored at hypothermic temperatures (4 h) is a key limiting factor for organ transplantation. This project focuses on an alternative preservation approach termed “partial freezing” which is based on achieving a thermodynamically stable, non-injurious frozen state. During extended preservation with partial freezing, hearts are particularly vulnerable to ice-mediated damage and metabolic perturbations but also warm ischemia.

In this project, we use zebrafish hearts as they can provide direct access to full complex organ structures, while being a much cheaper model to maintain and they allow high throughput screens that result in faster protocol improvements. The aim of this study is to leverage the zebrafish testbed to test if apoptosis inhibitors can rescue cardiac function after experiencing ischemic injury.

TA1_005. Preclinical whole organ and isolated tissue studies

Paul Iaizzo, UMN

Assessing toxic effects of CPAs on physiological function and subsequent tissue recovery in heart, lungs, heart/lung blocks, kidneys, and contractile tissues

One of the focus areas of our Visible Heart® Laboratories lies in developing critical platforms of reanimated organs or organ systems which we can use to assess physiological function and responses to pharmacological agents administered to modify function. These in vitro assessment platforms are ideal systems to study pharmacological pre-and post-treatments to minimize ischemic injury of organs prior to transplantation, CPA toxicity, cryopreservation methodologies, acute effects of administered adjuvants and/or the subsequent functionalities post-cryopreservation. This project focuses on assessing the toxic effects of CPAs on physiological function and subsequent tissue recovery with potential for clinical testing with large organ reanimations. The systems under study are heart, lungs, heart/lung blocks, kidneys, and contractile tissues.

TA1_006. Preservation of skin and vascular composite allografts

Korkut Uygun, MGH
Basak Uygun, MGH
Mehmet Toner, MGH
John Bischof, UMN
Michael Etheridge, UMN
Erik Finger, UMN

Develop de novo methods for preserving skin and VCAs to enable extended storage of these tissues in a viable state

Vascular composite allografts (VCAs), such as face and hand tissues, are characterized by multiple tissue types namely skin, muscle, fat, and bone. This heterogeneity offers unique challenges, ranging from immunogenicity of skin to complexity of adequate delivery of CPAs to the entire VCA. This project aims to develop methods (which currently do not exist) for preserving skin and VCAs which will enable extended storage of these tissues in a viable state. In the initial phase we worked on establishing a machine-perfusion protocol for VCA tissues, with limbs as the initial model. Secondary goals include development of novel biosensor approaches to detect rejection early and practically. This could further reduce or eliminate the need for immunosuppression and lead to the development of better preservation modalities that reduce rejection, enable tolerance induction, and develop self-immunomodulating grafts through genetic engineering. Notably, we have demonstrated successful supercooled preservation of rodent limbs for the first time.

TA1_007. Partial freezing of livers and liver MPS

Shannon Tessier, MGH
Mehmet Toner, MGH
Martin Yarmush, MGH
Korkut Uygun, MGH

Cryopreservation approach termed “partial freezing,” for thermodynamically stable, non-injurious frozen state at subzero

This project focuses on a new cryopreservation approach termed “partial freezing,” which has the potential to bank whole livers and liver MPS for weeks. Our nature-inspired approach is based on achieving a thermodynamically stable, non-injurious frozen state at subzero temperature ranging from -10 to -30°C, allowing the presence of ice and thereby avoiding the challenges of thermodynamically unstable supercooled storage at about -4°C or highly damaging cryogenic approaches below -120°C. Last year, our partial freezing multi-thermic protocol for rodent livers was used to successfully store rodent livers at -15°C for 5 days.This year, we addressed the limitations we identified in the multi-thermic perfusion modalities, such as coagulation and inflammatory events triggered through interaction of recirculating whole blood with the perfusion system that are not reflective of in vivo events. We incorporated a clinical grade oxygenator designed to create a more uniform blood flow, enhance gas transfer, and reduce pressure drops, whilst also reducing platelet adhesion and activation.