Estimation of absolute number of alveolar epithelial type 2 cells in mouse lungs: a comparison between stereology and flow cytometry

Abstract

Accurate estimation of the absolute number of a particular cell-type in whole organs is increasingly important in studies on organogenesis, and the remodelling and repair of diseased tissues. The unbiased estimation of the absolute number of cells in an organ is complicated, and design-based stereology remains the method of choice. This has led investigators to explore alternative approaches - such as flow cytometry - as a faster and less labour-intensive replacement for stereology. To address whether flow cytometry might substitute stereology, design-based stereology was compared with microfluorosphere-controlled flow cytometry, for estimation of the absolute number of alveolar epithelial type 2 cells (AEC2) in the lungs of two mouse strains: wild-type C57BL/6J mice and Sftpc-YFP mice. Using design-based stereology, ≈10.7 million and ≈9.0 million AEC2 were estimated in the lungs of wild-type C57BL/6J mice and Sftpc-YFP mice, respectively. Substantially fewer AEC2 were estimated using flow cytometry. In wild-type C57/BL6J mouse lungs, 59% of the AEC2 estimated by design-based stereology were estimated by flow cytometry (≈6.3 million), using intracellular staining for pro-surfactant protein C. Similarly, in Sftpc-YFP mouse lungs, 23% of the AEC2 estimated by design-based stereology were estimated by flow cytometry (≈2.1 million), using yellow fluorescent protein fluorescence. Our data suggest that flow cytometry underestimates AEC2 number, possibly due to impaired recoverability of AEC2 from dissociated lung tissue. These data suggest design-based stereology as the method of choice for the unbiased estimation of the absolute number of cells in an organ. LAY DESCRIPTION: There is much interest in studies on the pathological changes that accompany disease, to be able to count or estimate the number of a particular cell-type in solid tissue, such as an organ. The easiest way to do this is to make liquid suspensions of single cells from solid tissue, and then to count the number of cells of interest, using either a microscope, or automated cell counting (for example, a flow cytometer). Alternatively, solid tissue may be examined microscopically, where the cell-type of interest might also be counted 'by eye' or in an automated manner using software (called planimetry). All of these approaches to counting cells in solid organs come with serious drawbacks, and estimation of the cell number may thus be inaccurate. To overcome this, we have employed a combination of mathematical tools and statistical principles together with microscopy (called 'design-based stereology') that permits the unbiased counting of cells in microscopic fields, which can then be extrapolated to the entire solid tissue volume, to accurately estimate the number of a cell-type of interest in the solid tissue. We have compared this method with the estimation of cell number using a flow cytometer. Our data reveal that flow cytometry appreciably underestimates the total number of cells in solid tissue, where we used the lung as an example of solid tissue, and estimated the number of a unique cell-type in the lung: the alveolar epithelial type 2 cell, to compare stereology with flow cytometry. We believe that flow cytometry underestimates the cell number due to the difficulty of breaking up solid tissue into single cells, and being able to recover all of those single cells for analysis. Our data supports the recommendation to use stereology, not flow cytometry, to accurately estimate the number of a particular cell-type in solid tissue. Accurate estimation of the absolute number of a particular cell-type in whole organs is increasingly important in studies on organogenesis, and the remodelling and repair of diseased tissues. Although estimation of the relative number of cells might be straightforward, unbiased estimation of the absolute number of cells in an organ is complicated, and design-based stereology remains the method of choice. This has led investigators to explore alternative approaches - such as flow cytometry - as a faster and less labour-intensive replacement for stereology. To address whether flow cytometry might substitute stereology, design-based stereology was compared with microfluorosphere-controlled flow cytometry, for estimation of the absolute number of alveolar epithelial type 2 cells (AEC2) in the lungs of two mouse strains: wild-type C57BL/6J mice and Sftpc-YFP mice. Using design-based stereology, ≈10.7 million and ≈9.0 million AEC2 were estimated in the lungs of wild-type C57BL/6J mice and Sftpc-YFP mice, respectively. Substantially fewer AEC2 were estimated using flow cytometry. In wild-type C57/BL6J mouse lungs, 59% of the AEC2 estimated by design-based stereology were estimated by flow cytometry (≈6.3 million), using intracellular staining for pro-surfactant protein C. Similarly, in Sftpc-YFP mouse lungs, 23% of the AEC2 estimated by design-based stereology were estimated by flow cytometry (≈2.1 million), using yellow fluorescent protein fluorescence. Our data suggest that flow cytometry underestimates AEC2 number, possibly due to impaired recoverability of AEC2 from dissociated lung tissue. These data suggest design-based stereology as the method of choice for the unbiased estimation of the absolute number of cells in an organ.

Keywords: Alveolar epithelial type 2 cell; cell number; estimation; flow cytometry; physical fractionator; stereology.