Reply to Fujino et al.: Human Lung Cell Separation Strategies for Translational Research

From the Authors:

We thank Fujino and colleagues for their thoughtful letter regarding our publication (1). We are grateful to Fujino and colleagues for their interest in our publication and for their pioneering work in the development of what we have labeled the Col-Dis protocol (2). We acknowledge the strength of their Col-Dis protocol in the isolation of type II alveolar cells from lung tissue, generating the greatest proportion of epithelial cell adhesion molecule⁺ cytokeratin⁺ cells compared with all other protocols we tested, including the rapid collagenase-based dissociation protocol (Col-Short) featured in the publication. In contrast, our Col-Short protocol is characterized by a greater cell yield, minimal activation, no observed loss of protein markers, and a rapid processing time, making it a strong alternative to other protocols tested, for many applications.

We wish to expand on several points highlighted by Fujino and colleagues. First, we acknowledge that there was heterogeneity in the sample sizes across the different dissociation protocols. To address the concern that this may have introduced a selection bias that influenced our study results, we performed a reanalysis of the reported data using a repeated measures of variation statistical test after excluding groups with missing samples. In our new comparison of the Col-Long, Col-Short, and Col-Dis protocols with three diseased lungs (cases 5, 6, and 7) and three excess donor lungs (cases 8, 9, and 10), we obtained results similar to those reported in our publication. We still saw the highest cell yield from Col-Short compared with both the Col-Dis and Col-Long protocols (Figure 1A). Numbers of immune cell subsets were not different, although macrophage proportions were highest in Col-Long, similar to the original publication (Figure 1A). Col-Short had a low proportion of CD90⁺ fibroblasts and was outperformed by Col-Dis in acquiring a higher proportion of epithelial cells (Figure 1B). Because of the greater cell yield with Col-Short, the absolute numbers of fibroblasts and epithelial cells acquired through the Col-Short protocol were comparable to Col-Long and Col-Dis (Figure 1B).

Figure 1.

Comparison of major cell populations across Col-Long, Col-Short and Col-Dis dissociation protocols, assessed by flow cytometry. (A) Graphs showing the cell yield, proportion out of all CD45⁺ cells, and total cell count of major hematopoietic cell populations: macrophages, classical monocytes, NK cells, T cells, and B cells. (B) Graphs showing the proportion out of all CD45⁻ cells and total cell count of major nonhematopoietic cell populations: CD90⁺ fibroblasts, epithelial cells, and endothelial cells. Symbols denote six individual cases: Case 5 (inverted triangle), Case 6 (asterisk), Case 7 (star), Case 8 (circle), Case 9 (square) and Case 10 (triangle). Repeated measures ANOVA test with P < 0.05 is reported as “P =.” Error bars represent SEM.

We acknowledge that our study omitted the evaluation of type II alveolar cells. Indeed, the foremost purpose of the Col-Short protocol during its conception was the isolation of immune cells, hence our desire to use a collagenase-based approach to minimize surface protein cleavage, which we showed is an important issue for T cells and potentially also for macrophages in our publication. Parenchymal cells were not entirely omitted, because we identified CD90⁺ fibroblasts, endothelial cells, and, as a general population, epithelial cells from the single-cell suspensions generated with the Col-Short protocol. We did not assess any epithelial cell subsets, except for club cells. In future studies, we hope to investigate in greater detail subpopulations such as types I and II alveolar epithelial cells, ciliated cells, and basal cells.

Ultimately, we completely agree that one should tailor the selection of the tissue dissociation protocol to the specific research question at hand. Col-Short and Col-Dis, together with other protocols such as Lib-Elas (described by Travaglini and colleagues [3]), have unique advantages and disadvantages, which will appeal to different researchers on the basis of their cells of interest or experimental assays. Each dissociation protocol can impact the proportion and yield of cells, their activation state, and the presence of protein markers. Therefore, we wholeheartedly agree with Fujino and colleagues that a thoughtful approach that aligns the dissociation method with the experimental goals is key to any translational research in pulmonary biology and pathophysiology.