Cryobiophysical characteristics of genetically modified hematopoietic progenitor cells.
ABSTRACT 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.
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ABSTRACT: To explore the feasibility of ex vivo lymphocyte gene therapy for mild Hunter syndrome (mucopolysaccharidosis type II), we evaluated retrovirus-mediated gene transfer of the iduronate-2-sulfatase (IDS) coding sequence into peripheral blood lymphocytes from enzyme-deficient individuals (PBLMPS). Moloney murine leukemia virus-derived retroviral vectors were constructed by inserting the IDS cDNA under transcriptional regulation of the long terminal repeat (LTR) (in vector L2SN) or the cytomegalovirus (CMV) early promoter (vector LNC2). High-titer virus-producer cells were generated using amphotropic PA317 packaging cells. After 3 days of in vitro stimulation of T lymphocytes with anti-CD3 antibody and interleukin-2 (IL-2), PBLMPS were transduced once on each of the next 3 days. Seven to 21 days later, cultured PBLMPS were evaluated for gene transfer and IDS specific activity. Heterogeneous populations of L2SN-transduced PBLMPS had high levels of IDS enzyme activity (456 U/mg per hr +/- SD 292) despite a gene transfer efficiency of 5% or less. Owing to overexpression of IDS in that percentage of PBLMPS successfully transduced, IDS activity was increased above the deficiency found in patients with Hunter syndrome (< 20 U/mg per hr) to a level comparable with that of normal individuals (mean activity of uncultured normal leukocytes 807 U/mg per hr; SD 252). Reduced 35SO4-glucosaminoglycan (GAG) accumulation was observed in PBLMPS that had been transduced with L2SN, or when PBLMPS were grown in medium that had been "conditioned" by growth of L2SN-transduced cells. This latter result indicated that metabolic cross-correction occurred by means of intercellular enzyme transfer. These studies of retrovirus-mediated expression and metabolic correction, finding near-normal levels of IDS in cultured PBLMPS and metabolic correction, demonstrate the potential for treatment of mild, nonneuropathic Hunter syndrome by means of ex vivo lymphocyte gene therapy.Human Gene Therapy 02/1996; 7(3):283-90. · 4.02 Impact Factor
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ABSTRACT: Monocytes were isolated from fresh whole human blood and resuspended in Hanks balanced salt solution; a portion of the cells was mixed with an equal volume of 2M dimethyl sulfoxide (DMSO) to form a 1 M solution. Microliter volumes of cell suspension were placed directly onto a computer-controlled cryostage and cooled to a predetermined subzero temperature. Ice was nucleated in the extracellular medium and a continuous video record was made of the subsequent osmotically induced volume changes of individual cells owing to exposure to the concentrated extracellular solutes. Selected micrographs emphasizing the initial transient data were digitized for computer analysis with an interactive boundary tracing algorithm to determine metric parameters of specific cells, and apparent volume changes were measured as a function of elapsed time after nucleation. The Kedem-Katchalsky-coupled transport equations were fit to the data using a network thermodynamic model implemented on a microcomputer to determine values for the permeability properties Lp, omega, and sigma. Experiments were performed over the temperature range from -7 degrees to -10 degrees C. Cells pre-equilibrated with DMSO had a lower Lp and a higher activation energy, delta E, than without additive, although the statistical significance of the difference could not be substantiated. It was found that the movement of DMSO across the plasma membrane in response to extracellular freezing was apparently so much smaller than the water flux that values for omega and sigma could not be determined from the data base.Cryobiology 09/1991; 28(4):391-9. · 2.14 Impact Factor
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ABSTRACT: Cells can endure storage at low temperatures such as--196 degrees C for centuries. The challenge is to determine how they can survive both the cooling to such temperatures and the subsequent return to physiological conditions. A major factor is whether they freeze intracellularly. They do so if cooling is too rapid, because with rapid cooling insufficient cell water is removed osmotically to eliminate supercooling. Equations have been developed that describe the kinetics of this water loss and permit one to predict the likelihood of intracellular freezing as a function of cooling rate. Such predictions agree well with observations. Although the avoidance of intracellular freezing is usually necessary for survival, it is not sufficient. Slow freezing itself can be injurious. As ice forms outside the cell, the residual unfrozen medium forms channels of decreasing size and increasing solute concentration. The cells lie in the channels and shrink in osmotic response to the rising solute concentration. Prior theories have ascribed slow freezing injury to the concentration of solutes or the cell shrinkage. Recent experiments, however, indicate that the damage is due more to the decrease in the size of the unfrozen channels. This new view of the mechanism of slow freezing injury ought to facilitate the development of procedures for the preservation of complex assemblages of cells of biological, medical, and agricultural significance.The American journal of physiology 10/1984; 247(3 Pt 1):C125-42. · 3.28 Impact Factor