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Intracellular Flow Cytometric Measurement of Extracellular Matrix Components in Porcine Intervertebral Disc Cells

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Abstract

The objective of this study was to develop and demonstrate the utility of a novel method of evaluating intracellular levels of extracellular matrix (ECM) components in intervertebral disc (IVD) cells using flow cytometry. By using this method, this study discriminated between cell populations in porcine IVD and examined the response of IVD cells to monolayer cultures, a traditional method of cell expansion, by measuring phenotypic attributes of ECM component production. It was found that monolayer cultures affected collagen production of IVD cells while there were differences in collagen type II production between the cells isolated from the annulus fibrosus (AF) and nucleus pulposus (NP) regions of IVD. Size distributions of fresh and cultured cells were also presented while the relationships between cell size and intracellular collagen level revealed heterogeneous cell populations in AF and NP regions. Furthermore, this study showed that the intracellular collagen signals of IVD cells were significantly enhanced by the treatments of Brefeldin-A and ascorbic acid. This suggests that Brefeldin-A and ascorbic acid could be used to increase the sensitivity of flow cytometric analysis on intracellular collagen levels by maximizing collagen accumulation inside cells. Since a unique feature of the flow cytometric screening tool is the ability to discriminate between various cell populations in a single sample, the flow cytometric method developed in this study may have the potential to identify specific collagen-producing cell populations from tissues or cell cultures.

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Acknowledgments

The authors would like to thank Mr. Xiaoyi Li and Ms. Liza Laquian for their assistance with cell culture. This study was supported by Grant Number AR050609 from NIH (NIAMS).

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Authors and Affiliations

  1. Tissue Biomechanics Lab, Department of Biomedical Engineering, College of Engineering, University of Miami, P.O. Box 248294, Coral Gables, FL, 33124-0621, USA

    Daniel J. Flagler, Tai-Yi Yuan & Wei Yong Gu

  2. Stem Cell and Mechanobiology Lab, Department of Biomedical Engineering, University of Miami, Coral Gables, FL, USA

    Chun-Yuh Huang

  3. Department of Biology, University of Miami, Coral Gables, FL, USA

    Zhongmin Lu

  4. GRECC, Miami VA Medical Center, Miami, FL, USA

    Herman S. Cheung

  5. Department of Biomedical Engineering, University of Miami, Coral Gables, FL, USA

    Herman S. Cheung

Authors
  1. Daniel J. Flagler
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  2. Chun-Yuh Huang
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  4. Zhongmin Lu
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  5. Herman S. Cheung
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  6. Wei Yong Gu
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Correspondence to Wei Yong Gu.

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Flagler, D.J., Huang, CY., Yuan, TY. et al. Intracellular Flow Cytometric Measurement of Extracellular Matrix Components in Porcine Intervertebral Disc Cells. Cel. Mol. Bioeng. 2, 264–273 (2009). https://doi.org/10.1007/s12195-009-0045-y

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  • DOI: https://doi.org/10.1007/s12195-009-0045-y

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