# Extended limb preservation employing an optimization strategy for stabilization.

> **NIH NIH R43** · TISSUE TESTING TECHNOLOGIES, LLC · 2021 · $339,546

## Abstract

In collaboration with Prof. Brandacher at Johns Hopkins University we plan to improve upon the currently most
used clinical method of limb preservation, namely hypothermia at 0 to +4°C. We will build upon our base
preservation formulation, Unisol™, that has been shown to preserved whole large animals below +10°C for 6-
8h after total blood replacement with Unisol™ with normal functions upon return to physiological conditions.
We have also shown that Unisol™ can maintain blood vessel function for at least 6 days at both -7°C and +4°C
and that mouse hearts stored at 4°C for 18 hours in Unisol™ have a significantly faster return of heart function
than hearts stored in the gold standard hypothermic heart preservation solution Celsior (HTK). Encouraged by
these results we propose evaluation of Unisol supplemented with reagents targeting oxidation, apoptosis
(enhancers of stress tolerance) and metabolism (metabolic rate inhibition) in 2 specific aims using human
skeletal myoblasts and vascular endothelial cells to select optimal reagent concentrations in vitro and a rat
forelimb transplant model to evaluate the best supplement formulations developed in the in vitro studies. The
lead in vitro assay will be alamarBlue, however outcomes will be checked using alternative assays including
trypan blue, live/dead stain and MTT assay. Apoptosis will be evaluated if significant losses of metabolic
activity are observed 1 to 2 days after return to physiologic culture conditions. The in vivo studies will evaluate
the best formulations from the in vitro studies over 3 days of hypothermic storage by transplantation model.
Controls limbs will be preserved in Unisol™ and HTK. We anticipate that the supplemented formulation will
maintain viability and function of limbs for up to 36 hours, a considerable improvement over all current practice
methods. This innovation will not only increase storage time, it will also provide the opportunity for more closely
matched recipients and potentially induction of tolerance. Furthermore, we are improving on the most tried and
true method for hypothermic limb storage in clinical practice that is relatively inexpensive and easy to ship by
air. Demonstration of ≥24 hours of hypothermic storage with >70% retention of limb functions will be
considered to be a successful demonstration of feasibility for progression to a Phase II SBIR proposal for
further evaluation in large animal models and ex vivo human limb evaluation post-preservation.

## Key facts

- **NIH application ID:** 10257524
- **Project number:** 1R43HL158398-01
- **Recipient organization:** TISSUE TESTING TECHNOLOGIES, LLC
- **Principal Investigator:** Kelvin G.M. Brockbank
- **Activity code:** R43 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $339,546
- **Award type:** 1
- **Project period:** 2021-04-01 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10257524

## Citation

> US National Institutes of Health, RePORTER application 10257524, Extended limb preservation employing an optimization strategy for stabilization. (1R43HL158398-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10257524. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*