Cryobiophysical Characteristics of Genetically Modified Hematopoietic Progenitor Cells
Biomedical Engineering Center, University of Minnesota, Minneapolis, Minnesota 55455, USA. Cryobiology
(Impact Factor: 1.59).
04/1999; 38(2):140-53. DOI: 10.1006/cryo.1999.2157
The freezing responses of hematopoietic progenitor cells isolated from normal donors and from donors with mucopolysaccharidosis type I (MPS I) were determined using cryomicroscopy and analyzed using theoretical models for water transport and intracellular ice formation. The cells from donors with MPS I used in this investigation were cultured and transduced with a retroviral vector for the alpha-l-iduronidase (IDUA) enzyme in preclinical studies for human gene therapy. The water transport and intracellular ice formation (IIF) characteristics were determined at different time points in the culture and transduction process for hematopoietic progenitor cells expressing CD34 antigen from donors with MPS I and from normal donors. There were statistically significant changes in water transport, osmotically inactive cell volume fraction, and permeability between cells from different sources (normal donors vs donors with MPSI) and different culture conditions (freshly isolated vs cultured and transduced). Specifically, Lpg and Ea increased after ex vivo culture of the cells and the changes in permeability parameters were observed after as little as 3 days in culture. Similarly, the IIF characteristics of hematopoietic progenitor cells can also be influenced by the culture and transduction process. The IIF characteristics of freshly isolated cells from donors with MPS I were statistically distinct from those of cultured and transduced cells from the same donor. The ability to cryopreserve cells which are cultured ex vivo for therapeutic purposes will require an understanding of the biophysical changes resulting from the culture conditions and the manner in which these changes influence viability.
Available from: Willem Wolkers
- "The extent of mechanistic information that can be derived using these techniques, however, is limited. Cryomicroscopy can only be used to detect cell volume changes or intracellular ice formation   . Membrane properties are not measured directly. "
[Show abstract] [Hide abstract]
ABSTRACT: In order to predict optimal cooling rates for cryopreservation of cells, the cell-specific membrane hydraulic permeability and corresponding activation energy for water transport need to be experimentally determined. These parameters should preferably be determined at subzero temperatures in the presence of ice. There is, however, a lack of methods to study membrane properties of cells in the presence of ice. We have used Fourier transform infrared spectroscopy to study freezing-induced membrane dehydration of mouse embryonic fibroblast (3T3) cells and derived the subzero membrane hydraulic permeability and the activation energy for water transport from these data. Coulter counter measurements were used to determine the suprazero membrane hydraulic permeability parameters from cellular volume changes of cells exposed to osmotic stress. The activation energy for water transport in the ice phase is about three fold greater compared to that at suprazero temperatures. The membrane hydraulic permeability at 0 °C that was extrapolated from suprazero measurements is about five fold greater compared to that extrapolated from subzero measurements. This difference is likely due to a freezing-induced dehydration of the bound water around the phospholipid head groups. Using Fourier transform infrared spectroscopy, two distinct water transport processes, that of free and membrane bound water, can be identified during freezing with distinct activation energies. Dimethylsulfoxide, a widely used cryoprotective agent, did not prevent freezing-induced membrane dehydration but decreased the activation energy for water transport.
Biochimica et Biophysica Acta 03/2011; 1808(3):642-8. DOI:10.1016/j.bbamem.2010.11.021 · 4.66 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: An understanding of the osmotic response of cells during freezing and thawing is critical to the development of successful cryopreservation protocols. Calculation of osmotic parameters requires a detailed description of the cell volume. Two common ways to measure volume changes are to use optical measurement techniques or an electronic particle counter. The purpose of this investigation was to compare the effectiveness of these two techniques in measuring the osmotic response of human keratinocytes that were exposed to hypo- and hypertonic salt solutions. The osmotic parameters calculated using the two methods were not significantly different. Both the optical measurement method and the electronic particle counter have inherent advantages and disadvantages that must be considered when conducting osmotic studies.
Cryo letters 09/1999; 20(5):315-324. · 1.14 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: The freezing characteristics of genetically modified lymphocytes obtained from a donor with mucopolysaccharidosis 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, I(pg) and E(lp), for the cultured and transduced cells were determined to be 4.4 +/- 1.3 x 10(-14) m3/Ns and 173 +/- 25 kJ/mol, respectively. The IIF nucleation parameters, kappa and omega, were 5.5 x 10(10) K5 and 3.5 x 10(11) (l/m2 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.
Cell Transplantation 09/1999; 8(5):521-30. · 3.13 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.