| Research |
Biopreservation/Biothermodynamics
| Stabilization of Proteins and Membranes, Anhydrobiosis & Reversible Membrane Permeabilization |
| With the advances in medicine and biotechnology, especially in cell-based therapies, tissue engineering, regenerative medicine, gene therapy, cell transplantation and biopharmaceutical research, the demand for successful stabilization of proteins, cells and organs during storage is increasing. We believe that in order for the protein and cell-based therapies to be widely available, economical, efficient and safe, we need to devise ways to process, store, transport, and distribute these products without the requirement for cryogenic temperatures. We are interested in developing methods to stabilize and preserve proteins, cells, tissues and ultimately, organs in a desiccated/vitrified state. |
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 Biopreservation can be defined as the process of reversibly arresting the biochemical reactions and therefore the metabolism of an organism (in a state of suspended animation) in order to sustain function after prolonged exposure to otherwise lethal conditions. The state of the art for the preservation of most mammalian cells is cryopreservation, which requires processing at very low temperatures and storage in liquid nitrogen (Figure 1).
With the advances in medicine and biotechnology, especially in cell-based therapies, tissue engineering, regenerative medicine, gene therapy, cell transplantation and biopharmaceutical research, the demand for successful stabilization of proteins, cells and organs during storage is increasing. We believe that in order for the protein and cell-based therapies to be widely available, economical, efficient and safe, we need to devise ways to process, store, transport, and distribute these products without the requirement for cryogenic temperatures.
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 In nature, certain desiccation resistant organisms (such as Artemia cysts, escherichia coli, yeast and nematodes such as the tardigrade, see Figure 2) synthesize carbohydrates upon exposure to extreme temperatures and draught. Drying plant seeds and fungal spores also accumulate carbohydrates forming cytoplasmic glasses. This has been associated with their survival, fueling extensive research activity to utilize carbohydrates to preserve proteins, mammalian cells and tissues during freezing (cryopreservation), freeze-drying (lyophilization) or desiccation.
Following nature’s lead, we are interested in developing methods to stabilize and preserve proteins, cells, tissues and ultimately, organs in a desiccated/vitrified state. This is done by researching the mechanisms of desiccation damage for proteins and cells and devising methods to eliminate them. We are utilizing Fourier Transform Infrared (FTIR) Microscopy to detect changes in the mobility of water and the proteins during desiccation, freezing and storage. Our main goal is establishing the mechanisms of protection offered by the biopreservation agents in order to engineer efficient and safe preservation protocols. |
| It was established that for successful stabilization in the preserved state, carbohydrates (glucose, sucrose, raffinose and stachyose in plants, and trehalose in micro-organisms and animals) should be present on both sides of the cell membrane. This presents a problem since most of these carbohydrates are membrane impermeable. Various methods have been devised to reversibly permeabilize the membrane without destroying the cellular integrity. Currently applied methods are microinjection, thermal shock, electroporation, mechanical and acoustic exposure, endocytosis and transport through switchable membrane pores. Most of these methods have significant disadvantages that render them inefficient especially for large-scale processing (for example microinjection) for clinical applications. |
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We are therefore interested in developing alternative methods for the delivery of carbohydrates into live mammalian cells in large concentrations in an economical, fast and reliable way. One important point to note here is that most of the biopreservation solutions need to reach very high concentrations intracellularly (0.2M-10M, depending on the chemicals and the preservation method utilized). This presents a unique set of challenges. One method that is currently applied in our laboratory is electroporation, where a short duration electrical field is applied across the cell membrane for permeabilization. Another method currently being developed is reversible membrane stretching, where the permeability of a portion of a mammalian cell’s membrane is reversibly increased using osmotic shock. This enables large solutes to be loaded into the cell. In Figure 3, membrane bleb formation in an attached 3T3-S4 fibroblast in response to osmotic shock is shown. A fluorescent molecule is used to measure the increase in the intracellular mobility caused by the uptake of extracellular carbohydrate molecules. Note that increased fluorescence intensity shows increased cytoplasmic viscosity. Other loading methods currently investigated involve liposomal carriers, mechanical and thermal shock. |
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