Isolation and characterisation of alveolar type II pneumocytes from adult bovine lung

Abstract

Alveolar type II (ATII) cells play a key role as part of the distal lung epithelium, including roles in the innate immune response and as self-renewing progenitors to replace alveolar type I (ATI) cells during regeneration of the alveolar epithelium. Their secretion of surfactant protein helps to maintain homeostasis in the distal lung and exert protective, antimicrobial properties. Despite the cell's crucial roles, they remain difficult to study, in part due to inefficient and expensive isolation methods, a propensity to differentiate into alveolar type I cells in culture and susceptibility to fibroblast overgrowth from primary isolations. Published methods of isolation often require specialist technology, negatively impacting the development of in vitro models of disease, including bovine tuberculosis (BTB), a serious re-emerging disease in both animals and humans worldwide. We present here a simple and cost-effective method that may be utilised in the generation of bovine primary ATII cells. These exhibit an ATII phenotype in 2D and 3D culture in our studies and are conducive to further study of the role of ATII cells in bovine respiratory diseases.

Figure 1

Schematic of the purification procedure from animal to culture. (a) Lungs were obtained directly from slaughter and taken back to the laboratory, where aseptic dissection took place. (b) The tissue was then washed repeatedly with DPBS containing EDTA and penicillin/streptomycin. (c) Tissue was digested at 37 °C, then filtered sequentially through mesh of decreasing pore sizes. (d) The filtrate was overlaid onto bovine IgG-coated bacterial grade petri dishes to remove fibroblasts and macrophages. (e) Non-adherent cells were then loaded in 4% Percoll™ onto a Percoll™ gradient, overlaying onto 10–30% Percoll, which generated an ATII enriched emulsion at the 10–30% interface. (f) These cells were then washed, counted and plated out in SAGM, containing 100 U/mL penicillin/streptomycin.

Figure 2

Isolation was performed from the distal lobe of the right lung. (a) H&E staining (see methods) was used to verify the health status and phenotype of the region dissected. Multiple alveolar ducts are marked by arrows. (b) The enriched fraction obtained by Percoll gradient, with some residual fibroblast contamination (arrows). (c) Representative image of the cobblestone morphology observed in isolated ATII cells. (d) Cells isolated in the pellet fraction of the Percoll gradient, including macrophages and erythrocytes, with negligible epithelial content. (e) Percoll fraction image acquired using a 40 x objective, highlighting a macrophage (black arrow) and convex erythrocyte (red arrow). Images representative of three independent isolations.

Figure 3

IF analysis of isolated cells cultured on 8-chamber slides over a period of 48 hours. (a) The cell surface marker CD74, (b) cytoplasmic cytokeratin 18 (CK18) and (c) surfactant protein SPC are specific to the ATII phenotype, whilst epithelial cell adhesion molecule (EpCAM) (d) is a general identifier of the epithelial cell phenotype. Images representative of three independent isolations.

Figure 4

Three dimensional (3D) culture of isolated cells on permeable inserts and embedded in Matrigel. (a) Isolated ATII cells were seeded onto Matrigel-coated (1:10 dilution) Transwell™ 12 mm inserts and cultured for 2 days submerged, followed by 8 days at air-liquid interface. (b) H&E staining of two week old organoid cross-sections containing necrotic (N) material, with live peripheral cells staining strongly for large spherical vesicles (arrows), consistent with those containing surfactant. (c) Representative TEM image, as carried out on cross sections of cells cultured on 6 mm Greiner Thincerts, confirming presence of lamellar bodies. (d) Ringed structures surrounding the ‘lumen’ in isolated cells cultured as a 3D suspension. Images representative of three independent isolations.

Figure 5

Comparative analysis of gene expression, comparing SFTPC (ATII marker) with that of AQP5 (ATI marker). Expression of each gene was normalised to the housekeeping control, GAPDH. Statistical analysis found a significant reduction in SFTPC between 72 and 120 h, with further reduction observed at 168 h. An inverse relationship was seen between SFTPC and AQP5 over time, with a concurrent increase in AQP5 at the same time points. Mean ± SD, n = 6 (across two experiments). *P ≤ 0.05; **P ≤ 0.001; ***P ≤ 0.0001 (two tailed t test, unpaired).

Figure 6

Removal of contaminating fibroblasts from cultures of isolated ATII epithelial cells. (a) (i) Fibroblasts, residual from the isolation process, are highlighted by arrows. Cells subjected to HBSS treatment for a minimum of 30 minutes, result in an adherent and non-adherent phenotype. (ii) Cobblestone morphology exhibited by adherent cells, indicative of epithelial cells. (iii) Non-adherent cells with a spindle-like morphology, indicative of fibroblast enrichment. (b) IF staining with the ATII marker Pro SP-C and co-stain with CD90, a surface marker previously identified in both epithelial and fibroblast cultures; adherent cells are positive for both markers, whilst non-adherent cells appear negative for Pro SP-C expression. Images representative of three treatments, each of 30 minutes duration.