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: A new model was developed to predict transmembrane water transport and diffusion-limited ice formation in cells during freezing without the ideal-solution assumption that has been used in previous models. The model was applied to predict cell dehydration and intracellular ice formation (IIF) during cryopreservation of mouse oocytes and bovine carotid artery endothelial cells in aqueous sodium chloride (NaCl) solution with glycerol as the cryoprotectant or cryoprotective agent. A comparison of the predictions between the present model and the previously reported models indicated that the ideal-solution assumption results in under-prediction of the amount of intracellular ice at slow cooling rates (<50 K/min). In addition, the lower critical cooling rates for IIF that is lethal to cells predicted by the present model were much lower than those estimated with the ideal-solution assumption. This study represents the first investigation on how accounting for solution nonideality in modeling water transport across the cell membrane could affect the prediction of diffusion-limited ice formation in biological cells during freezing. Future studies are warranted to look at other assumptions alongside nonideality to further develop the model as a useful tool for optimizing the protocol of cell cryopreservation for practical applications.Journal of Applied Physics 04/2014; 115(14):144701-144701-13. DOI:10.1063/1.4870826 · 2.19 Impact Factor
Article: Greening Sample Treatments[Show abstract] [Hide abstract]
ABSTRACT: Remote sensing, noninvasive and direct analysis of samples cannot be performed in any analytical situation and, unfortunately, in many cases the greening of a procedure involves only the reduction of the side effects of sample treatments. The evolution of alternative green sample preparation tools pursues reduction of the amount of sample, reduction or elimination of organic solvents, simultaneous multiclass compound extraction and potential for automation and/or high-throughput determination.In this chapter, the state-of-the-art of green alternatives for sample treatment have been discussed, based on the classification of the different alternatives according to the physical state of the samples, solids or liquids and also considering the generation of a gas or vapor phase directly from the samples. Many different objectives like analyte isolation from the matrix, analyte enrichment and/or sample clean-up and selective isolation of target analytes will be considered here.Comprehensive Analytical Chemistry 01/2011; 57:87-120. DOI:10.1016/B978-0-444-53709-6.00005-7
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ABSTRACT: Background aims. Current methods of mesenchymal stromal cell (MSC) cryopreservation result in variable post-thaw recovery and phenotypic changes caused by freezing. The objective of this investigation was to determine the influence of ex vivo cell expansion on phenotype of MSCs and the response of resulting phenotypes to freezing and thawing. Methods. Human bone marrow aspirate was used. MSCs were isolated and cells were assessed for total count, viability, apoptosis and senescence over 6 passages (8-10 doublings/passage) in ex vivo culture. One half of cells harvested at each passage were replated for continued culture and the other half were frozen at 1 degrees C/min in a controlled-rate freezer. Frozen samples were stored in liquid nitrogen, thawed and reassessed for total cell count, viability and senescence immediately and 48 h after thaw. Results. Viability did not differ significantly between samples before freeze or after thaw. Senescence increased over time in pre-freeze culture and was significantly higher in one sample that had growth arrest both before freeze and after thaw. Freezing resulted in similar initial post-thaw recovery in all samples, but 48-h post-thaw growth arrest was observed in the sample with high senescence only. Conclusions. High pre-freeze senescence appears to correlate with poor post-thaw function in MSC samples, but additional studies are necessary to obtain a sample sizes large enough to quantify results.Cytotherapy 10/2014; 17(1). DOI:10.1016/j.jcyt.2014.06.008 · 3.10 Impact Factor