# Deep Supercooling of Liver Cells and 2D and 3D Tissue Constructs: Effect of Attachment

> **NIH NIH R21** · MASSACHUSETTS GENERAL HOSPITAL · 2020 · $210,000

## Abstract

Summary
 Apart from the preservation of a “whole human” there are three outstanding questions in the field of
preservation 1) Can we preserve whole organs to reduce quality and logistics issues in transplantation where
over 120000 patients are currently waiting for a replacement organ? 2) Can we devise an effective preservation
scheme to ease the dissemination for the rapidly growing market of engineered tissue products? 3) Can we
preserve primary cells with metabolic and enzymatic activity similar to fresh cells robustly and cheaply? Finally,
one can ask the question: Can we answer these three questions with a “unified method”? Currently, the clinical
gold standard for organ preservation is static cold storage (+4 oC) with up to 72 hours for kidney and ideally 12
hours for liver which is limiting in terms of logistics and flexibility. On the other end of the spectrum,
cryopreservation and vitrification - despite successes at the cell level for some cell types - do not project success
at the organ and tissue level soon. This current technological gap necessitates a superior and unified
biopreservation method that is designed from the ground up with the long-term goal of storage of cells,
tissues/tissue products and organs beyond one week to relieve the current logistic constraints. Based on our
recent published works, we propose that non-freezing (supercooling) preservation (SCP) of living matter provides
such a solution that bridges the gap between preservation of cells, tissue and organs. Our short-term goal is to
develop a novel “Deep Supercooling” (DSC) preservation methods starting with single cells and multicellular
tissue constructs. The intermediate-term goal, following up this exploratory period, is then to apply these to a)
storage of commercial tissue-constructs, and b) successful organ storage from kidneys to hearts and livers.
 Supercooling can be achieved for small samples (~<1ml) at temperatures (-4 to -6 oC) for ~7 days at most
without any freezing as in our earlier work. Yet, it has been impossible to keep a large volume (10s of milliliters)
of preservation solution at very low temperatures (~-20 oC) for long times (~100 days) in a practical manner.
Recently we addressed this in a breakthrough, dubbed “Deep Supercooling (DSC) via Surface Sealing”,
where we seal water with an immiscible liquid (oils, alkanes, alcohols) in a solid container to achieve a practically
stable supercooling temperature (down to -20 oC) for large volumes (up to 100 ml) for long period (up to 100
days). The objective of the proposed study is thus to develop “Deep Supercooling” preservation method for 3
models of hepatic cells/tissues (suspended cells, 2D plated cells, 3D spheroids) and then compare the long-term
(5 day) success of these 3 models. Deep supercooling at ultralow (-10 to -20 oC) temperatures can drastically
slow down metabolism and injury processes compared to cold storage (at 4 oC) and we expect this will
significantly improve storage time an...

## Key facts

- **NIH application ID:** 10020997
- **Project number:** 5R21GM136002-02
- **Recipient organization:** MASSACHUSETTS GENERAL HOSPITAL
- **Principal Investigator:** Osman Berk USTA
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $210,000
- **Award type:** 5
- **Project period:** 2019-09-20 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10020997

## Citation

> US National Institutes of Health, RePORTER application 10020997, Deep Supercooling of Liver Cells and 2D and 3D Tissue Constructs: Effect of Attachment (5R21GM136002-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10020997. Licensed CC0.

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