Freezing Characteristics of Genetically Modified Lymphocytes for the Treatment of MPS II

Abstract

The freezing characteristics of genetically modified lymphocytes obtained from a donor with mucopolysac charidosis type II (MPS II) were determined using cryomicroscopy and controlled rate freezing studies to determine postthaw viability. The cells from a donor with MPS II used in this investigation were cultured and transduced with a retroviral vector for the iduronate-2-sulfatase (IDS) enzyme for clinical studies for human gene therapy. The water transport and intracellular ice formation (IIF) characteristics of the cells were determined after completion of the culture and transduction protocol. The water transport parameters, L m and £, p , for the cultured and transduced cells were determined to be 4.4 ± 1.3 x 10~1 4 m 3 /Ns and 173 ± 25 kJ/mol, respectively. The IIF nucleation parameters, K and Q, were 5.5 x 10 10 K 5 and 3.5 x 10" (1/m 2 s), respectively. The postthaw 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 thawed under the same conditions. These studies are essential in understanding the biophysical changes resulting from the ex vivo culture of cells and the manner in which these changes influence the ability of the cells to be cryopreserved.

Modeling the interaction of biological cells with a solidifying interface

“…In the base case, a particle was placed 76.3 lm in front of an interface with an initial perturbation, x = 2 cos 0.12py, in a domain of size 200 lm · 200 lm. The initial radius of the particle was 7.5 lm, the internal concentration within the cell was 0.020 M, and the cell membrane permeability constant was set to 1.5 · 10 4 lm 3 /N s, which is the accepted value for the lymphoblastoid cells used in the cryomicroscopy experiments [22]. The Hamaker constant was set to 0.5g cell lm/s 2 where g cell represents the average mass of a cell.…”

Clinical mesenchymal stromal cell products undergo functional changes in response to freezing

“…Ex vivo culture of cells can also influence freezing response. Both hematopoietic progenitors and lymphocytes exhibited changes in subzero water transport and intracellular ice formation tendencies after ex vivo culture, 9,10 which in turn can influence freezing response. Francois and colleagues quantified diminished response for indoleamine 2,3-dioxygenase (critical to immunomodulatory cell function) for frozen and thawed MSCs when compared to fresh non-frozen cells.…”

Effect of Cryopreservation on Autologous Chimeric Antigen Receptor T Cell Characteristics

“…21 Consequently, the biophysical and physiologic properties of thawed mature cells cultured ex vivo may vary from those for freshly isolated cells that encounter the same stressors. 22,23 To better understand the clinical impact of these phenomena, we studied relevant phenotypic and functional cellular alterations during and after the manufacture of CARTs cryo-thawed at either or both ends of cell manufacturing.…”