Cryobiophysical characteristics of genetically modified hematopoietic progenitor cells

The freezing responses of hematopoietic progenitor cells isolated from norm al donors and from donors with mucopolysaccharidosis type I(MPS I) were det ermined using cryomicroscopy and analyzed using theoretical models for wate r transport and intracellular ice formation. The cells from donors with MPS I used in this investigation were cultured and transduced with a retrovira l vector for the alpha-L-iduronidase (IDUA) enzyme in preclinical studies f or human gene therapy. The water transport and intracellular ice formation (IIF) characteristics were determined at different time points in the cultu re and transduction process for hematopoietic progenitor cells expressing C D34 antigen from donors with MPS I and from normal donors. There were stati stically significant changes in water transport, osmotically inactive cell volume fraction, and permeability between cells from different sources (nor mal donors vs donors with MPST) and different culture conditions (freshly i solated vs cultured and transduced). Specifically, L-pg and E-a increased a fter ex vivo culture of the cells and the changes in permeability parameter s were observed after as little as 3 days in culture. Similarly, the IIF ch aracteristics of hematopoietic progenitor cells can also be influenced by t he culture and transduction process. The LIF characteristics of freshly iso lated cells from donors with MPS were statistically distinct from those of cultured and transduced cells from the same donor, The ability to cryoprese rve cells which are cultured ex vivo for therapeutic purposes will require an understanding of the biophysical changes resulting from the culture cond itions and the manner in which these changes influence viability. (C) 1999 Academic Press.