Freezing characteristics of genetically modified lymphocytes for the treatment of MPS II

The freezing characteristics of genetically modified lymphocytes obtained f rom a donor with mucopolysaccharidosis type II (MPS II) were determined usi ng cryomicroscopy and controlled rate freezing studies to determine posttha w viability. The cells from a donor with MPS II used in this investigation were cultured and transduced with a retroviral vector fbr the iduronate-2-s ulfatase (IDS) enzyme for clinical studies for human gene therapy. The wate r transport and intracellular ice formation (IIF) characteristics of the ce lls were determined after completion of the culture and transduction protoc ol. The water transport parameters, L-pg and E-lp, for the cultured and tra nsduced cells were determined to be 4.4 +/- 1.3 x 10(-14) m(3)/Ns and 173 /- 25 kJ/mol, respectively. The IIF nucleation parameters, kappa and Omega, were 5.5 x 10(10) K-5 and 3.5 x 10(11) (1/m(2) s), respectively. The postt haw viability of the genetically modified cells was less than the viability of the freshly isolated cells from the same donor. The postthaw viability of the cultured and transduced cells from a donor with MPS II was also less than that observed with cells from a normal donor that were frozen and tha wed under the same conditions. These studies are essential in understanding the biophysical changes resulting from the ex vivo culture of cells and th e manner in which these changes influence the ability of the cells to be cr yopreserved.