Graduate Program

Biological Sciences

Degree Name

Master of Science (MS)

Semester of Degree Completion

2013

Thesis Director

Michael A. Menze

Thesis Committee Member

Gary A. Bulla

Thesis Committee Member

Andrew S. Methven

Abstract

Anhydrobiotic organisms such as the brine shrimp Artemia franciscana have developed strategies to survive and thrive under conditions of limited water availability. One strategy is the accumulation of late embryo genesis abundant (LEA) proteins. Cells of Drosophila melanogaster (Kc167), which had previously been genetically modified to express a group 1 LEA protein (LEA1.3) found in A. franciscana, were used to test the impact of water stress due to freezing to -80°C. Two different freezing devices were used to determine the impact of the freezing protocol on the viability of cells after freezing and thawing. The first method used a 12-well microplate insulated in a novel passive freezing device produced by BioCision (Larkspur, CA) and the second method used traditional cryogenic vials. The membrane integrity of cells after freezing and thawing was compared between cells expressing LEA1.3 (Kc167-LEA1.3) and control cells that lack the transgenic protein (Kc167).

Furthermore, two different freezing solutions were employed. Efficiency of cryopreservation was compared between a standard freezing solution composed of cell culture medium (M3) and a novel choline-lactobionate based freezing buffer (ASFB), both supplemented with dimethyl sulfoxide (DMSO). There was a significantly higher amount of cells with intact membranes for Kc167-LEA1.3 cells than Kc167 wild type cells I both ASFB and M3 medium (n = 12, P < 0.05). This correlates with a higher rate of viability for cells expressing the AfLEA1.3 protein. Also, a significantly higher numbers of cells with intact membranes was found for both cell lines when frozen in 12-well plates as opposed to the freezing in 2 ml cryogenic vials (n = 24, P < 0.05). To ensure a steady freezing rate, two passive freezing devices were used. Plate based freezing in multi-well plates may help to improve the experimental procedures utilized in cell storage and cell based bioassays. The expression of AfLEA1.3 protein and the use of a plate-based freezing device yields higher viability for cells exposed to extreme low temperatures.

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