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Quantitative Assay for Quality Assurance of Human Cells for Clinical Transplantation

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Transplantation of human cells after isolation and culture has become an important alternative for treatment of acute or chronic skin wounds. To increase the efficacy and reduce cost for transplantation of skin cells, more efficient and accurate techniques for evaluation of cell proliferation are needed. Hemocytometer counts provide a valid assessment of cell proliferation and viability, but they are very labor intensive and require removal of the cells from their substrate. In this study, hemocytometer counts were compared with a fluorometric assay (n = 21 per condition) that uses the commercially available reagent alamarBlueTM, which is reduced to a fluorescent substrate by cellular dehydrogenases. Human epidermal keratinocytes were inoculated at 200, 600, 2000, and 6000 cells/cm2 incubated for 6 days in modified MCDB 153 medium. Alamar BlueTM was incubated with cells for 2 h at 37°C, and fluorescence was measured with a microplate reader at 590 nm. Hemocytometer counts (×10−4) from the respective cell inoculation densities were 0.30 ± 0.04, 1.07 ± 0.10, 6.37 ± 0.62, and 16.99 ± 0.96. Fluorescence values (×10−3) for the respective inoculation densities were 0.14 ± 0.01, 0.34 ± 0.02, 1.20 ± 0.09, and 1.79 ± 0.12. Regression analysis showed a statistical significant (p < 0.0001) correlation (r 2 = 0.87) between cell counts and optical density from the alamarBlueTM assay. These data demonstrate that alamarBlueTM provides a valid substitute for cell counts to assess cell proliferation before clinical transplantation of engineered skin. AlamarBlueTM also allows repeated, nondamaging assessment of living cells over time. These advantages are expected to increase the validity and reliability of quality assurance standards for transplanted skin cells, and to increase the efficacy of healing of cutaneous wounds.
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Keywords: Burns; Chronic wounds; Cultured keratinocytes; Skin substitutes; Wound healing

Document Type: Research Article

Affiliations: 1: Departments of Surgery and Biomedical Engineering, University of Cincinnati, Cincinnati, OH, USA, Research Department, Shriners Burns Hospital, Cincinnati, OH, USA 2: Departments of Surgery and Biomedical Engineering, University of Cincinnati, Cincinnati, OH, USA

Publication date: 2006-02-01

More about this publication?
  • Cell Transplantation publishes original, peer-reviewed research and review articles on the subject of cell transplantation and its application to human diseases. To ensure high-quality contributions from all areas of transplantation, separate section editors and editorial boards have been established. Articles deal with a wide range of topics including physiological, medical, preclinical, tissue engineering, and device-oriented aspects of transplantation of nervous system, endocrine, growth factor-secreting, bone marrow, epithelial, endothelial, and genetically engineered cells, among others. Basic clinical studies and immunological research papers are also featured. To provide complete coverage of this revolutionary field, Cell Transplantation will report on relevant technological advances, and ethical and regulatory considerations of cell transplants. Cell Transplantation is now an Open Access journal starting with volume 18 in 2009, and therefore there will be an inexpensive publication charge, which is dependent on the number of pages, in addition to the charge for color figures. This will allow work to be disseminated to a wider audience and also entitle the corresponding author to a free PDF, as well as prepublication of an unedited version of the manuscript.

    Cell Transplantation is now being published by SAGE. Please visit their website for the most recent issues.

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