Defined conditions for long-term expansion of murine and human alveolar epithelial stem cells in three-dimensional cultures

Abstract

Alveolar type 2 cells (AT2s) serve as stem cells of the alveoli and restore cell numbers after injury. Here, we describe a detailed protocol for the isolation, purification, and culture of murine and human AT2s. We have developed chemically defined and stroma-free culture conditions that enable expansion and maintenance of AT2s. The culture conditions are scalable and compatible with high-throughput chemical and genetic screenings and can potentially be used to generate large AT2 numbers for cell-based therapies. For complete details on the use and execution of this protocol, please refer to Katsura et al. (2020).

Keywords: Cell Biology; Cell culture; Cell isolation; Organoids; Stem Cells.

Graphical abstract

Figure 2

Single-cell suspension and FACS sorting of murine AT2c (A) Visualization of lung single-cell suspension. (B) Gates for FACS sorting of AT2 cells. (C) Validation of AT2s purity using cytospin preparations of sorted cells and staining with SFTPC (green). The yellow arrow indicates SFTPC negative cell. Scale bar:50 μm. (D) Quantification of SFTPC⁺ in total DAPI⁺ cells. Data are presented as mean ± SEM. n=3.

Figure 1

Dissection and dissociation of murine lung (A) Schematic representation of crucial steps of murine lung dissection and dissociation to single cells suspension. (B) Images illustrate murine lung before and after perfusion and inflation with digestion solution. (C) Images illustrate steps after lung removal, separated lobes and lung mincing. Scale bar: 5 mm. (D) Examples of lung dissociation at indicated incubation time with digestion solution. (E) Visualization of cell pellet before and after red blood lysis buffer.

Figure 3

Mincing and dissociation of human lung (A) Visualization of human lung before and after pleura removal. Scale bar: 10 mm. (B) Illustration of lung piece cut out for dissection. Blue circle depicted airway and vasculature that were dissected before lung mincing. Scale bar: 10 mm. (C) Images taken at different steps of lung dissociation. (D) Validation of human AT2s purity using cytospin preparations of HTII-280⁺ sorted cells and staining with HTII-280 (green) and SFTPC (red). The yellow arrow points out on SFTPC negative cell. Scale bar:50 μm. (E) Quantification of SFTPC⁺ in total DAPI⁺ cells. Data are presented as mean ± SEM. n=3.

Figure 4

Alveolospheres plating and expansion (A) Schematic illustration of alveolar culture preparation and plating. (B) Example of matrigel droplets containing AT2 cells. (C) Mouse alveolospheres cultured for 10–12 days. (D) Human alveolospheres cultured for 12–14 days. Scale bar: 500 μm and 50 μm.

Figure 5

Validation of alveolospheres culture (A) Schematic representation of fixation and embedding of mouse alveolospheres. (B) Images showing indicated step of mouse alveolospheres embedding for cryosection. (C) Schematic illustration of human alveolospheres collection following fixation. (D) Images showing steps for human alveolospheres embedding. (E) Immunostaining with AT2 cell marker -SFTPC, SFTPC and HTII-280 and AT1 cell marker - AGER (gray) on sections collected from mouse (left) and human (right) alveolospheres. Scale bar: 100 and 30 μm.