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.

Isolation of Alveolar Type II Cells from Adult Bovine Lung

“…This differentiation is in part reversible in vitro by culturing the ATII cells in Matrigel (100%; BD Biosciences) or on Matrigel‐coated permeable membranes as polarized monolayers. Under these conditions, expression of the ATII markers CD74 and proSP‐C is retained (Lee et al., ; Wang et al., ), along with the formation of lamellar inclusion bodies, the hallmark identifier of ATII cells in vitro and in vivo (Dobbs, Pian, Maglio, Dumars, & Allen, ; Lee et al., ; Mao et al., ). Thus, if the ATII phenotype is lost during routine culture of cell stocks, a subculture can be grown as outlined above to assess phenotype.…”

“…ATII cells can also be cultured as submerged 2D monolayers prior to studies performed on Transwell inserts. Although the ATII phenotype is lost in the former, it can be resurrected in the latter, as evidenced by immunofluorescence and transmission electron microscopy (Lee et al., ). The reversibility of differentiation is not a new discovery.…”

“…This is removed and resuspended in small airway growth medium to dilute the Percoll and allow pelleting by centrifugation. Finally, the cell pellet is resuspended and used to establish cultures and determine phenotype by immunofluorescent staining of ATII cell markers (Lee et al, 2018). 3.…”

“…The reason for this is the detrimental effect of enzymatic digestion upon cell surface markers. The binding of debris to the ATII marker CD74 in both FACS and magnetic bead isolation has been reported (Lee et al, 2018). Changing the recipe of the digestion mix is not advised, due to the high content of collagen previously reported in bovine lungs (Cohen, Zelikoff, & Schlesinger, 2000) and the improved yield as a result of the inclusion of trypsin (Dobbs et al, 1986).…”

“…The full protocol combines dissection, mincing, and enzymatic digestion (see Basic Protocol 1), selective adherence (see Basic Protocol 2), and gradient centrifugation (see Basic Protocol 3). This approach results in a predominantly epithelial cell population (85-90%) that exhibits an ATII phenotype (Lee et al, 2018) together with 10-15% fibroblasts. The latter may be removed for downstream applications by cold trypsinization (see Basic Protocol 4).…”