Title page for ETD etd-11152005-124419

Type of Document Master's Thesis
Author Li, Guanglei
Author's Email Address gli1@lsu.edu
URN etd-11152005-124419
Title Cryopreservation of Reproductive Cells and Tissues
Degree Master of Science in Mechanical Engineering (M.S.M.E.)
Department Mechanical Engineering
Advisory Committee
Advisor Name Title
Ram Devireddy Committee Chair
Harris Wong Committee Member
Tryfon T. Charalampopoulos Committee Member
  • water transport
  • supzero cooling
  • cryopreservation
  • differential scanning calorimeter
  • membrane permeability
  • optimal rate of freezing storage
  • Krogh model
Date of Defense 2005-11-07
Availability unrestricted
Cryopreservation of reproductive cells and tissues is a useful technique to deliver intact genomes, maximize the distribution of favorable genes, control disease spread. It is widely applied to many fields such as biology, medicine, ecology and agriculture. To optimize the cryopreservation procedure, the biophysical responses of reproductive tissues at different cooling conditions and in different mediums must be clearly understood. Here we studied the biophysical response during freezing of: 1) bovine sperm frozen at a cooling rate of 20 ˚C/min in three media: without cryopreservation agents(CPAs), with 0.7 M glycerol, and with 0.7 M glycerol along with 1.5 mg/ml of cholesterol-loaded cyclodextrin (CLC); 2) equine ovarian tissue cooled either at 40˚C/min or at 0.5˚C/min from 25˚ to 4˚C, and then cooled to subzero temperatures at 5 ˚C/min in the presence and absence of 0.85 M glycerol and 0.85 M dimethylsulfoxide; and 3) Macaca mulatta ovarian tissue cooled at 0.5˚C/min or 40˚C/min from 25˚ to 4˚C, and then cooled to subzero temperatures at 5 ˚C/min in the absence and presence of 0.85 M glycerol, 0.85 M dimethylsulfoxide and 0.85 Methylene glycol. For all the three reproductive systems, a shape-independent Differential Scanning Calorimeter (DSC) technique was used to measure the volumetric shrinkage during freezing. A model of water transport was then fitted to the experimentally obtained volumetric shrinkage data and the best fit membrane permeability parameters (Lpg and ELp) was yielded. For bovine sperm, Lpg ranged from 0.02 to 0.036 Ám/min-atm and ELp ranged from 26.4 to 42.1 kcal/mol. The subzero water transport parameters of bovine sperm are significantly different from those reported in literature. The experimentally determined optimal rate of freezing bovine spermatozoa agrees quite closely with the theoretically calculated range, from 45 to 60 ˚C/min. For equine tissue samples, Lpg ranged from 0.06 to 0.73 Ám/min-atm and ELp ranged from 6.1 to 54.2 kcal/mol. For rhesus ovarian tissue Lpg ranged from 0.7 to 0.15 Ám/min-atm and ELp ranged from 22.1 to 32.1 kcal/mol. Data obtained for equine and macaque ovarian tissue data suggest that the optimal cryopreservationn rates are significantly dependent upon suprazero cooling conditions and choice of cryoprotective agent.
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