Our overall goal is to develop long-term storage strategies that can be used for tissue engineering, cell transplantation, and stem cell applications. 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. Dry storage provides a long-term preservation strategy that alleviates many of the problems associated with cryopreservation: (1) dried storage is the only preservation method that permits the ambient temperature, long-term storage of cells, significantly simplifying the distribution of the therapeutic product; (2) the dry product is much lighter than conventionally preserved frozen products; (3) dry storage is an accepted method for the manufacturing of therapeutic products by regulatory authorities; and (4) the significantly lower concentrations and """"""""non-toxic"""""""" nature of stabilizers used in dry storage (-0.2M) compared to freeze-thaw (1.5-2M) or vitrification (approximately 6-8M) protocols reduce the need for removal of these agents before injection, transfusion, or transplantation. Our hypothesis is that molecular mobility in the desiccated state is the primary cause of cellular injury during dried storage and that stabilizers such as trehalose are needed to halt or minimize molecular mobility in order to achieve stability and long-term viability of cells in a dried state. We will test our hypothesis and generate fundamental understanding of the behavior and stability of murine embryonic stem (ES) cells in desiccated state in four distinct, but interactive Specific Aims.
In Specific Aim 1, we will investigate the molecular mobility and glass formation within desiccated mouse ES cells in the presence and absence of various sugars.
In Specific Aim 2, we will genetically engineer mouse ES cells to synthesize trehalose in order to achieve intracellular glassy state in dry state and thus reduce molecular mobility.
In Specific Aim 3, we will probe the molecular mechanisms of cell death in desiccated storage and explore the use of molecular inhibitors to prevent cell death after drying.
In Specific Aim 4, we will investigate the stability of dried mouse ES cells and optimize the desiccation and rehydration conditions most conducive to the differentiation of dry preserved mouse ES cells into hepatocytes.
| He, Xiaoming; Park, Eric Y H; Fowler, Alex et al. (2008) Vitrification by ultra-fast cooling at a low concentration of cryoprotectants in a quartz micro-capillary: a study using murine embryonic stem cells. Cryobiology 56:223-32 |
| Risco, Ramon; Elmoazzen, Heidi; Doughty, Marshal et al. (2007) Thermal performance of quartz capillaries for vitrification. Cryobiology 55:222-9 |
