Our overall goal is to develop long-term hepatocyte storage strategies that can be used for the treatment of liver failure. Although cell stasis is routinely achieved in nature by anhydrobiotic organisms through desiccation at ambient temperatures, the only existing strategy for long-term storage of mammalian cells is cryopreservation. Cryopreservation relies on cryogenic temperatures (typically < -80 degrees C) in order to halt all chemical reactions that result in cell death during storage. On the other hand, the pharmaceutical industry has made significant strides in storing proteinaceous drugs, liposomes, membranes, and viral particles in dry state using various small sugar molecules as stabilizers. Sugars, such as disaccharides, enter glassy phase at ambient temperatures at low moisture levels (approximately 5%) and minimize molecular mobility. Our hypothesis is that molecular mobility in the desiccated state needs to be completely prevented to stop deterioration of the cells and the cellular injury during dried storage. Stabilizers such as sugars, therefore, are needed to halt molecular mobility in order to achieve stability and long-term viability of cells in a dried state. We will test our hypothesis is four distinct but interactive Specific Aims.
In Specific Aim #1, we will determine molecular mobility in dried cells in the glassy and near glassy state.
In Specific Aim #2, we will investigate the mechanisms of cell death associated with cellular desiccation and to develop strategies to modulate these processes to improve survival.
In Specific Aim #3, we will determine the stability of desiccated living cells and the key parameters affecting shelf life.
In Specific Aim #4, we will test the efficacy of desiccated cells in small animal models of liver failure. The proposed studies will be carried out using several key sugars including trehalose, sucrose, raffinose, stachyose, and mixtures thereof. With the advancements being made in tissue engineering, cell transplantation, stem cell biology, and gene therapy, the clinical demand for effective long-term storage methods for cells and tissues will continue to increase for many different tissue types, including liver. Desiccation of mammalian cells is a very attractive alternative strategy that has the potential to truly disseminate these powerful technologies to medical centers, hospitals, and physicians' offices.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK046270-13
Application #
7037634
Study Section
Surgery and Bioengineering Study Section (SB)
Program Officer
Doo, Edward
Project Start
1994-01-01
Project End
2008-03-31
Budget Start
2006-04-01
Budget End
2008-03-31
Support Year
13
Fiscal Year
2006
Total Cost
$226,067
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02199
Hand, Steven C; Denlinger, David L; Podrabsky, Jason E et al. (2016) Mechanisms of animal diapause: recent developments from nematodes, crustaceans, insects, and fish. Am J Physiol Regul Integr Comp Physiol 310:R1193-211
Weng, Lindong; Tessier, Shannon N; Smith, Kyle et al. (2016) Bacterial Ice Nucleation in Monodisperse D2O and H2O-in-Oil Emulsions. Langmuir 32:9229-36
Podrabsky, Jason E; Hand, Steven C (2015) Physiological strategies during animal diapause: lessons from brine shrimp and annual killifish. J Exp Biol 218:1897-906
Hand, Steven C; Menze, Michael A (2015) Molecular approaches for improving desiccation tolerance: insights from the brine shrimp Artemia franciscana. Planta 242:379-88
Heo, Yun Seok; Nagrath, Sunitha; Moore, Alessandra L et al. (2015) ""Universal"" vitrification of cells by ultra-fast cooling. Technology (Singap World Sci) 3:64-71
Abazari, Alireza; Meimetis, Labros G; Budin, Ghyslain et al. (2015) Engineered Trehalose Permeable to Mammalian Cells. PLoS One 10:e0130323
Abazari, Alireza; Chakraborty, Nilay; Hand, Steven et al. (2014) A Raman microspectroscopy study of water and trehalose in spin-dried cells. Biophys J 107:2253-62
Borcar, Apurva; Menze, Michael A; Toner, Mehmet et al. (2013) Metabolic preconditioning of mammalian cells: mimetic agents for hypoxia lack fidelity in promoting phosphorylation of pyruvate dehydrogenase. Cell Tissue Res 351:99-106
Patil, Yuvraj N; Marden, Brad; Brand, Martin D et al. (2013) Metabolic downregulation and inhibition of carbohydrate catabolism during diapause in embryos of Artemia franciscana. Physiol Biochem Zool 86:106-18
Chakraborty, Nilay; Menze, Michael A; Elmoazzen, Heidi et al. (2012) Trehalose transporter from African chironomid larvae improves desiccation tolerance of Chinese hamster ovary cells. Cryobiology 64:91-6

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