Document Detail


High-throughput single cell arrays as a novel tool in biopreservation.
MedLine Citation:
PMID:  19303403     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Microwell array cytometry is a novel high-throughput experimental technique that makes it possible to correlate pre-stress cell phenotypes and post-stress outcomes with single cell resolution. Because the cells are seeded in a high density grid of cell-sized microwells, thousands of individual cells can be tracked and imaged through manipulations as extreme as freezing or drying. Unlike flow cytometry, measurements can be made at multiple time points for the same set of cells. Unlike conventional image cytometry, image analysis is greatly simplified by arranging the cells in a spatially defined pattern and physically separating them from one another. To demonstrate the utility of microwell array cytometry in the field of biopreservation, we have used it to investigate the role of mitochondrial membrane potential in the cryopreservation of primary hepatocytes. Even with optimized cryopreservation protocols, the stress of freezing almost always leads to dysfunction or death in part of the cell population. To a large extent, cell fate is dominated by the stochastic nature of ice crystal nucleation, membrane rupture, and other biophysical processes, but natural variation in the initial cell population almost certainly plays an important and under-studied role. Understanding why some cells in a population are more likely to survive preservation will be invaluable for the development of new approaches to improve preservation yields. For this paper, primary hepatocytes were seeded in microwell array devices, imaged using the mitochondrial dyes Rh123 or JC-1, cryopreserved for up to a week, rapidly thawed, and checked for viability after a short recovery period. Cells with a high mitochondrial membrane potential before freezing were significantly less likely to survive the freezing process, though the difference in short term viability was fairly small. The results demonstrate that intrinsic cell factors do play an important role in cryopreservation survival, even in the short term where extrinsic biophysical factors would be expected to dominate. We believe that microwell array cytometry will be an important tool for a wide range of studies in biopreservation and stress biology.
Authors:
Kenneth L Roach; Kevin R King; Korkut Uygun; Steven C Hand; Isaac S Kohane; Martin L Yarmush; Mehmet Toner
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Publication Detail:
Type:  Evaluation Studies; Journal Article; Research Support, N.I.H., Extramural     Date:  2009-03-19
Journal Detail:
Title:  Cryobiology     Volume:  58     ISSN:  1090-2392     ISO Abbreviation:  Cryobiology     Publication Date:  2009 Jun 
Date Detail:
Created Date:  2009-05-12     Completed Date:  2009-10-22     Revised Date:  2014-09-14    
Medline Journal Info:
Nlm Unique ID:  0006252     Medline TA:  Cryobiology     Country:  United States    
Other Details:
Languages:  eng     Pagination:  315-21     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Cell Survival
Cryopreservation / methods*
Hepatocytes / metabolism
Membrane Potentials
Tissue Array Analysis / methods*
Grant Support
ID/Acronym/Agency:
K99 DK080942/DK/NIDDK NIH HHS; P41 EB002503/EB/NIBIB NIH HHS; P41 EB002503/EB/NIBIB NIH HHS; P41 EB002503-05/EB/NIBIB NIH HHS; R01 DK043371/DK/NIDDK NIH HHS; R01 DK046270/DK/NIDDK NIH HHS; R01 DK046270/DK/NIDDK NIH HHS; R01 DK046270-15/DK/NIDDK NIH HHS; R01 GM071345/GM/NIGMS NIH HHS; R01AI063795/AI/NIAID NIH HHS
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