Title page for ETD etd-11112009-142047

Type of Document Master's Thesis
Author Guha, Avishek
Author's Email Address aguha1@tigers.lsu.edu
URN etd-11112009-142047
Title Intracellular Ice Formation in Adult Stem Cells in the Presence of Polyvinyl Pyrrolidone
Degree Master of Science in Mechanical Engineering (M.S.M.E.)
Department Mechanical Engineering
Advisory Committee
Advisor Name Title
Devireddy, Ram Committee Chair
Monroe, William Todd Committee Member
Park, Sunggook Committee Member
  • ice nucleation
  • water transport
  • cryomicroscopy
Date of Defense 2009-10-21
Availability restricted
The main objective of this work was to assess the effect of 10% (w/v) polyvinylpyrrolidone (PVP) on the pattern of intracellular ice formation (IIF) in human adipose tissue derived adult stem cells (ASCs) in the absence of serum and other cryoprotective agents (CPAs). Passage 1 (P1) ASCs were cultured, washed and suspended in either 1x PBS (Phosphate Buffered Saline) or 10% w/v solution of PVP in 1x PBS. The freezing experiments were carried out using a fluorescence microscope equipped with a Linkam™ cooling stage using two different temperature/time cooling protocols. Both the cooling protocols had a common cooling ramp: cells were cooled from 20 ˚C to –8 ˚C at 20 ˚C/min and then further cooled to –13 ˚C at 1 ˚C/min (during which the extra-cellular medium froze very rapidly and was accompanied by the formation of intracellular ice in ~96% of the cells, as noted by visible "flashing/darkening"). At this point we employed either, cooling protocol 1: the cells were cooled from –13 ˚C to – 40 ˚C at a pre-determined cooling rate of 1, 5, 10, 20 or 40 ˚C/min and then thawed back to 20 ˚C at 20 ˚C/min; or cooling protocol 2: the cells were re-warmed from –13 ˚C to –5 ˚C at 20 ˚C/min and then re-cooled at a pre-determined rate of 1, 5, 10, 20 or 40 ˚C/min to –40 ˚C. Almost all (>95%) of the ASCs frozen in 1x PBS and protocol 1 exhibited IIF whereas almost none (<5%) of the ASCs frozen in 1x PBS and protocol 2 exhibited IIF. The lack of IIF in cells cooled in 1x PBS and protocol 2 was due to the initial loss of cell viability (confirmed through an additional membrane dye exclusion study) that was associated with the formation of IIF in the common cooling ramp, described earlier. Similarly, almost all (>95%) of the ASCs frozen in 10% PVP in PBS and protocol 1 exhibited IIF where as ~0, ~40, ~47, ~67 and ~100% of the ASCs frozen in 10% PVP in PBS and protocol 2 exhibited IIF at a cooling rate of 1, 5, 10, 20 or 40 ˚C/min, respectively. The observed increase in the % of ASCs exhibiting IIF when frozen in 10% PVP and protocol 2, is presumably due to PVP mitigating the damaging effects of IIF during the common cooling ramp.
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